Assays for measuring binding kinetics and binding capacity of acceptors for lipophilic or amphiphilic molecules

ABSTRACT

Aspects of the invention relate to methods for measuring the binding constant of a lipophilic or amphiphilic molecule acceptor for a lipophilic or amphiphilic molecule. Methods involve rapid, cell-free competition assays including a labeled lipophilic or amphiphilic molecule and nanoparticle.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/073,941, entitled “ASSAYS FOR MEASURING BINDING KINETICS AND BINDINGCAPACITY OF ACCEPTORS FOR LIPOPHILIC OR AMPHIPHILIC MOLECULES” filedMar. 18, 2016 which claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application Ser. No. 62/134,788, entitled “ASSAYS FORMEASURING BINDING KINETICS AND BINDING CAPACITY OF ACCEPTORS FORLIPOPHILIC OR AMPHIPHILIC MOLECULES” filed on Mar. 18, 2015, which isherein incorporated by reference in its entirety.

FIELD OF INVENTION

The present invention relates generally to the use of nanoparticles inassays for monitoring the binding of lipophilic or amphiphilicmolecules.

BACKGROUND OF THE INVENTION

Coronary heart disease, which can manifest as heart attacks or suddendeath from lethal arrhythmias, is the number one killer in the U.S. andworldwide. Accurate estimation of cardiovascular disease risk is acritical first step in applying life-saving preventative therapies tohigh risk populations. State-of-the-art methods to ascertain risk,however, remain imperfect. Evidence shows that measuring Cholesterolefflux capacity of a certain human serum fraction significantly enhancesprediction accuracy of cardiovascular risk. Measurement of an aspect ofReverse Cholesterol Transport (RCT) with an assay that involves culturedcells and human serum samples improves the accuracy of clinical riskassessment for heart disease (Khera et al. (2011) N. Engl. J. Med.364:127). However, because this assay entails tissue culture techniques,radiolabeled cholesterol, and a 72-hour turnaround time, its clinicalutility is limited.

SUMMARY OF THE INVENTION

Quantifying cholesterol transport in complex biological matrices iscritically important because of the central role cholesterol balance oroverload plays in many human pathologies, such as heart disease (Lusis(2000) Nature 407:233). Cholesterol is a hydrophobic molecule minimallysoluble in the aqueous milieu of biological systems, yet it is found inmillimolar quantities in blood, owing to solubilization in lipidcarriers known as lipoproteins which bind and transport cholesterol.Despite the important role cholesterol-lipoprotein interactions have inunderstanding cholesterol transport, the dissociation constant (K_(D))of cholesterol from lipoprotein-containing serum fractions remainsunknown. Of particular interest is the strength of cholesterolinteractions with high density lipoproteins (HDL), as these structuresmediate cholesterol clearance from peripheral tissues, a process termedReverse Cholesterol Transport (RCT) (Rosenson et al. (2012) Circulation125:1905). A simple, automatable assay measuring serum affinity forlipophilic or amphiphilic molecules, such as cholesterol is highlydesirable.

Disclosed herein is a nanoparticle-enabled, rapid and cell-freecompetition assay which directly measures the affinity of lipophilic oramphiphilic molecule acceptors, including human serum. This method wasused to discover the dissociation constant (K_(D)) of serum and a serumfraction enriched in HDL for cholesterol.

Aspects of the invention relate to a method for measuring theequilibrium constant of an acceptor for a molecule, comprising:providing a lipophilic or amphiphilic molecule, wherein the molecule canprovide a detectable signal; providing a structure, the structurecomprising a nanostructure core and a lipid layer surrounding andattached to the nanostructure core, wherein the structure quenches thesignal of the molecule when the structure and the molecule areproximate; providing an acceptor; allowing the acceptor to compete withthe structure for binding with the molecule; and measuring the signal,wherein the level of the signal correlates with the equilibrium constantof the acceptor for the molecule.

In certain embodiments, the method further comprises increasing theamount of acceptor provided. In certain embodiments, the method furthercomprises increasing the amount of structure provided.

In certain embodiments, increasing the amount of acceptor provided leadsto an increase in signal. In certain embodiments, increasing the amountof structure provided leads to a decrease in signal. In certainembodiments, increasing or decreasing the amount of molecule improvesthe signal to noise ratio of the system.

In certain embodiments, the method is a component of an assay. Incertain embodiments, the method is a component of a diagnostic assay. Incertain embodiments, the method is a method for assessing cardiovascularrisk in a subject.

Other aspects of the invention relate to a kit for measuring anequilibrium constant of an acceptor for a molecule, comprising: alipophilic or amphiphilic molecule, wherein the molecule can provide adetectable signal; and a structure, the structure comprising ananostructure core and a lipid layer surrounding and attached to thenanostructure core, wherein the structure quenches the signal of themolecule when the structure and the molecule are proximate.

In certain embodiments, the kit further comprises instructions for useof the kit for measuring the equilibrium constant of the acceptor forthe molecule.

Other aspects of the invention relate to a system for measuring anequilibrium constant of an acceptor for a molecule, comprising: asample, the sample comprising: a lipophilic or amphiphilic molecule,wherein the molecule can provide a detectable signal; a structure, thestructure comprising a nanostructure core and a lipid layer surroundingand attached to the nanostructure core, wherein the structure quenchesthe signal of the molecule when the structure and the molecule areproximate; an acceptor; and a detector for measuring the signal.

In certain embodiments, the system described further comprises a deviceconfigured to calculate the equilibrium constant of the acceptor for themolecule based on the detected signal. In certain embodiments, thesystem further comprises a radiation source configured to induce thesignal.

In certain embodiments of the method, kit, or system herein described,the lipid layer is a bilayer. In certain embodiments of the method, kitor system herein described, the molecule is a steroid or a derivative oranalog thereof.

In certain embodiments of the method, kit, or system herein described,the molecule is a lipopolysaccharide or a derivative or analog thereof.In certain embodiments of the method, kit, or system herein described,the molecule is a steroid or a derivative of analog thereof. Severalnon-limiting classes of steroids include Cholestanes, Cholanes,Pregnanes, Androstanes and Estranes or derivative or analog thereof.

In certain embodiments, of the method, kit, or system herein described,the molecule is BODIPY-cholesterol. In certain embodiments of themethod, kit, or system herein described, the signal is fluorescence. Incertain embodiments of the method, kit, or system herein described, thesignal is fluorescence polarization.

In certain embodiments of the method, kit, or system herein described,the nanostructure core is an inorganic material. In certain embodimentsof the method, kit, or system herein described, the nanostructure coreis a metal. In certain embodiments of the method, kit, or system hereindescribed, the nanostructure core is gold.

In certain embodiments of the method, kit, or system herein described,the structure further comprises apolipoprotein bound to at least theouter surface of the lipid layer. In certain embodiments of the method,kit, or system herein described, the apolipoprotein is apolipoproteinA-I, apolipoprotein A-II, or apolipoprotein E.

In certain embodiments of the method, kit, or system herein described,the acceptor is a lipoprotein. In certain embodiments of the method,kit, or system herein described, the acceptor is a high-densitylipoprotein (HDL). In certain embodiments of the method, kit, or systemherein described, the acceptor is a component of serum. In certainembodiments of the method, kit, or system herein described, the serum ishuman serum. In certain embodiments of the method, kit, or system hereindescribed, the serum is enriched for HDL. In certain embodiments of themethod, kit, or system herein described, the serum is depleted for ApoB.

The subject matter of this application involves, in some cases,interrelated products, alternative solutions to a particular problem,and/or a plurality of different uses of structures and compositions.

Other advantages and novel features of the present invention will becomeapparent from the following detailed description of various non-limitingembodiments of the invention when considered in conjunction with theaccompanying figures. In cases where the present specification and adocument incorporated by reference include conflicting and/orinconsistent disclosure, the present specification shall control. If twoor more documents incorporated by reference include conflicting and/orinconsistent disclosure with respect to each other, then the documenthaving the later effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

Non-limiting embodiments of the present invention will be described byway of example with reference to the accompanying figures, which areschematic and are not intended to be drawn to scale. In the figures,each identical or nearly identical component illustrated is typicallyrepresented by a single numeral. For purposes of clarity, not everycomponent is labeled in every figure, nor is every component of eachembodiment of the invention shown where illustration is not necessary toallow those of ordinary skill in the art to understand the invention. Inthe figures:

FIG. 1 shows an exemplary binding experiment used to measure binding andkinetics of a cholesterol acceptor, in this case ApoB-depleted humanserum. Baseline fluorescence intensity was first recorded in a 6.25 uMsolution of BODIPY cholesterol in 100 ul of 20% ethanol in water. Next100 ul of 500 nM HDL AuNP was added and fluorescence intensity wasmeasured for 30 minutes at 2 minute read intervals. At this point theconcentrations of BODIPY cholesterol and HDL AuNP were 312.5 nM and 250nM, respectively. Finally, cholesterol acceptor was added andfluorescence was measured over 60 minutes. FIG. 1 shows measurement ofApoB-depleted serum at final assay concentrations of 0.00%, 0.16%,0.32%, 0.49%, and 0.65%, respectively (addition of 0, 5, 10, 15, and 20ul of ApoB-depleted human serum prepared as described above added to the200 ul volume containing BODIPY cholesterol and HDL AuNP). The amount offluorescence recovery rises with the increasing amounts of cholesterolacceptor added. Data points are the mean of wells plated in triplicate.

FIG. 2 shows a lipidated nanoparticle templated with 5 nm diameter goldwhich was titrated into 250 nM of fluorescent BODIPY-Cholesterol.Fluorescence quenching was measured upon binding. Nonlinear regressionanalysis modeling the BODIPY-Cholesterol interaction with the lipidatednanoparticle as a multiple binding site interaction with same K_(D) andno cooperativity yields a K_(D) of 40±9 nM and a binding site number of16±1 binding sites. Error bars denote standard deviations of technicalquadruplicates.

FIG. 3 shows serum or ApoB-depleted serum that was titrated into asolution of 20 nM NP and 125 nM BODIPY-Cholesterol. The effective K_(D)of serum in this case was calculated as 1.4×10⁻⁴, and of ApoB-depletedserum as 3.0×10⁻⁴. Error bars denote standard deviations of technicalquadruplicates.

FIG. 4 shows an illustration of lipidated nanoparticles synthesis.

FIG. 5 shows the manipulate function in Mathematica that was used toassess the performance and plausibility of the function generated fromsolving a system of binding equations. This allowed for assessment offunction performance as well as manual estimation of initial startingparameters for KD.

FIG. 6 shows an assay measuring patient serum affinity for cholesterolbinding. The data show a correlation between the performance of theassay of the invention and a currently accepted assay. The normalizedefflux assay is plotted on the x-axis, and can be seen to vary inmagnitude among participants from approximately 0.8 to approximately1.4, approximately 1.0 to approximately 2.0, or approximately 0.8 toapproximately 2.4 with a value of 1.0 being equivalent to the effluxvalue of the pooled sample.

DETAILED DESCRIPTION

The invention is based, at least in part, on the development of a rapid,cell-free competition assay for measuring binding between lipophilic oramphiphilic molecules and a lipophilic or amphiphilic molecule acceptor,such as serum or fractions thereof. Methods described herein, usinglabeled lipophilic or amphiphilic molecules, such as cholesterolanalogs, and a lipidated nanoparticle, are rapid and high throughput.These novel assays have widespread applications including: assessingrisk of disorders, such as cardiovascular disorders, by measuringlipophilic or amphiphilic molecule binding in patient samples;conducting screens for agents that influence lipophilic or amphiphilicmolecule efflux; investigating lipophilic or amphiphilic moleculebinding kinetics and binding capacity; and investigating small moleculebinding and binding capacity to HDLs (e.g., competition assays).

Aspects of the invention relate to a competition assay that can detectthe K_(D) of lipophilic or amphiphilic molecule acceptors, such as humanserum, for lipophilic or amphiphilic molecules, such as cholesterol.When the assay is applied to a sample, such as a human serum sample, itcan be used to assess the strength of the interaction between lipophilicor amphiphilic molecules, such as cholesterol, and high densitylipoproteins (HDL) within the serum sample. HDL plays a significant rolein clearance of cholesterol from peripheral tissues, in a processreferred to as Reverse Cholesterol Transport (RCT). Assessing thebinding capacity of HDL to cholesterol in a serum sample from a subjectprovides valuable information on the subject's functional capacity forRCT.

Novel assays described herein have multiple advantages over previousmethods for assessing RCT, including: i) Assays described herein areautomatable and involve simple and straightforward pipetting steps thatcan be programmed into laboratory hardware/robotic liquid handlingsystems. A high-throughput—96- or 384-well format can allow for dozensof samples to be read and processed simultaneously; ii) Noradiochemicals need to be used in these assays. Radiolabelledcholesterol, used in previously described assays had to be handled in aspecific manner and had to be disposed of carefully. Moreover, mostclinical laboratories do not have facilities to carry out radiometrictests; iii) Assays described herein are very rapid—owing to the rapidbinding kinetics of both natural lipophilic or amphiphilic moleculeacceptors and materials described herein, this test can be performed,including incubation times, within hours with minimal human input. Bycontrast, previously described Cholesterol efflux assays used inresearch take 4 days from start to finish; iv) very low serum volumesare required—assays described herein require a serum input on the orderof 100 ul from ordinary blood draw tubes. Furthermore, given thestability of lipophilic or amphiphilic molecules, such as cholesterolacceptors in serum, this test has the potential to be done as an“add-on” or “reflex” to routinely collected specimens ordered for otherpurposes.

Methods described herein involve binding between a lipidatednanoparticle and a labeled lipophilic or amphiphilic molecule, such as acholesterol analog. As used herein, a “lipidated nanoparticle” refers toa nanoparticle and that is associated with one or more lipids. Lipidatednanoparticles are described further in, and incorporated by referencefrom PCT/US2009/002540, entitled “Nanostructure Suitable forSequestering Cholesterol and Other Molecules.”

It should be appreciated that any lipophilic or amphiphilic moleculescan be compatible with aspect of the invention. As used herein, alipophilic molecule refers to a molecule that can dissolve in fats,oils, lipids, and non-polar solvents. Examples of lipophilic groupsinclude, but are not limited to, cholesterol, a cholesteryl or modifiedcholesteryl residue, adamantine, dihydrotesterone, long chain alkyl,long chain alkenyl, long chain alkynyl, olely-lithocholic, cholenic,oleoyl-cholenic, palmityl, heptadecyl, myrisityl, bile acids, cholicacid or taurocholic acid, deoxycholate, oleyl litocholic acid, oleoylcholenic acid, glycolipids, phospholipids, sphingolipids, isoprenoids,such as steroids, vitamins, such as vitamin E, fatty acids eithersaturated or unsaturated, fatty acid esters, such as triglycerides,pyrenes, porphyrines, Texaphyrine, adamantane, acridines, biotin,coumarin, fluorescein, rhodamine, Texas-Red, digoxygenin,dimethoxytrityl, t-butyldimethylsilyl, t-butyldiphenylsilyl, cyaninedyes (e.g. Cy3 or Cy5), Hoechst 33258 dye, psoralen, or ibuprofen. Thecholesterol moiety may be reduced (e.g. as in cholestan) or may besubstituted (e.g. by halogen). A combination of different lipophilicgroups in one molecule is also possible. The lipophilic molecule may bea sterol, such as cholesterol.

As used herein, an amphiphilic molecule refers to a molecule thatpossesses both hydrophilic and lipophilic properties. Severalnon-limiting examples of amphiphilic compounds include phospholipids,cholesterol, glycolipids, fatty acids, bile acids, saponins and localanaesthetics.

In some embodiments, the molecule is a steroid or a derivative or analogthereof. As used herein, a steroid refers to an organic compound thatcontains four cycloalkane rings that are joined to each other. Severalnon-limiting examples of classes of steroids include Cholestane,Cholane, Pregnane, Androstane or Estrane. In some embodiments, themolecule is a lipopolysaccharide or a derivative or analog thereof. Asused herein a lipopolysaccharide refers to a molecule consisting of alipid and a polysaccharide joined by a covalent bond.

The lipophilic or amphiphilic molecules associated with aspects of theinvention produce a detectable signal. In some embodiments, thelipophilic or amphiphilic molecule is labeled with a fluorescent label.The terms “fluorescent label”, “fluorescent dye”, and “fluorophore” areused interchangeably herein to refer to moieties that absorb lightenergy at a defined excitation wavelength and emit light energy at adifferent wavelength. Examples of fluorescent labels include, but arenot limited to: Alexa Fluor dyes (Alexa Fluor 350, Alexa Fluor 488,Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660 and Alexa Fluor 680), AMCA, AMCA-S,BODIPY dyes (BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY493/503, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589,BODIPY 581/591, BODIPY 630/650, BODIPY 650/665), Carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), Cascade Blue, Cascade Yellow, Coumarin 343,Cyanine dyes (Cy3, Cy5, Cy3.5, Cy5.5), Dansyl, Dapoxyl,Dialkylaminocoumarin, 4′,5′-Dichloro-2′,7′-dimethoxy-fluorescein,DM-NERF, Eosin, Erythrosin, Fluorescein, FAM, Hydroxycoumarin, IRDyes(IRD40, IRD 700, IRD 800), JOE, Lissamine rhodamine B, Marina Blue,Methoxycoumarin, Naphthofluorescein, Oregon Green 488, Oregon Green 500,Oregon Green 514, Pacific Blue, PyMPO, Pyrene, Rhodamine B, Rhodamine6G, Rhodamine Green, Rhodamine Red, Rhodol Green,2′,4′,5′,7′-Tetra-bromosulfone-fluorescein, Tetramethyl-rhodamine (TMR),Carboxytetramethylrhodamine (TAMRA), Texas Red, Texas Red-X,5(6)-Carboxyfluorescein, 2,7-Dichlorofluorescein,N,N-Bis(2,4,6-trimethylphenyl)-3,4:9,10-perylenebis(dicarboximide, HPTS,Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTO 465, ATTO 488, ATTO 495, YOYO-1,5-FAM, BCECF,dichlorofluorescein, rhodamine 110, rhodamine 123, YO-PRO-1, SYTOXGreen, Sodium Green, SYBR Green I, Alexa Fluor 500, FITC, Fluo-3,Fluo-4, fluoro-emerald, YoYo-1 ssDNA, 2′,4′,5′,7′-Tetra-bromosulfone 1dsDNA, YoYo-1, SYTO RNASelect, Diversa Green-FP, Dragon Green, EvaGreen,Surf Green EX, Spectrum Green, NeuroTrace 500525, NBD-X, MitoTrackerGreen FM, LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation),WEGFP (post-activation), FlASH-CCXXCC, Azami Green monomeric, AzamiGreen, green fluorescent protein (GFP), EGFP (Campbell Tsien 2003), EGFP(Patterson 2001), Kaede Green,7-Benzylamino-4-Nitrobenz-2-Oxa-1,3-Diazole, Bex1, Doxorubicin, LumioGreen, and SuperGlo GFP. Those of ordinary skill in the art will know ofother suitable fluorescent labels for the assays described herein, orwill be able to ascertain such, using routine experimentation.

In some embodiments, the fluorescent label is from thedifluoro-boraindacene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene)family (BODIPY) family. In some embodiments, the labeled lipophilic oramphiphilic molecule is BODIPY-Cholesterol, NBD-Cholesterol ordansyl-Cholesterol or their associated cholesteryl esters. In otherembodiments, fluorescent cholesterol analogues that possess intrinsicfluorescence (e.g., dehydroergosterol and cholestatrienol) and those inwhich a fluorophore or photoreactive group is attached (e.g.,NBD-Cholesterol, BODIPY-Cholesterol, and dansyl-cholestanol).

Nanoparticles described herein, such as lipidated gold nanoparticles,have the ability to quench fluorescence emitted by a fluorescentlylabeled lipophilic or amphiphilic molecules when the fluorescentlylabeled lipophilic or amphiphilic molecule is in close proximity to thenanoparticle. Accordingly, in assays described herein, binding betweenthe nanoparticle and the fluorescently labeled lipophilic or amphiphilicmolecule leads to a reduction in the levels of lipophilic or amphiphilicmolecule measured in a sample.

Aspects of the invention involve lipophilic or amphiphilic moleculeacceptors. As used herein, a “lipophilic or amphiphilic moleculeacceptor” refers to a molecule that can bind to a lipophilic oramphiphilic molecule. A lipophilic or amphiphilic molecule acceptor maybe involved in transporting a lipophilic or amphiphilic molecule, suchas cholesterol, to the liver from peripheral tissues. A lipophilic oramphiphilic molecule acceptor used in assays described herein can beserum, such as human serum. The serum can be enriched for HDLs. In someembodiments, the serum is depleted for Apolipoprotein B (ApoB). Additionof a lipophilic or amphiphilic molecule acceptor, such as a cholesterolacceptor, to assays described herein causes competition for cholesterolbinding with the nanoparticle and causes the fluorescent signal torecover.

As used herein, an acceptor refers to a molecule that binds cholesterol.Examples of an acceptor includes, but it is not limited to,apolipoprotein A1 (ApoA1), high density lipoprotein (HDL), albumin,serum, including human serum, or apolipoprotein B (ApoB)-depleted humanserum.

Measurement of fluorescence in assays described herein, followingaddition of a cholesterol acceptor can be used to assay levels of HDL ina sample and to determine binding and kinetics of a cholesterolacceptor. Since a low HDL level in serum can correlate with an increasedrisk of disorders associated with cholesterol, such as cardiovasculardisorders, assays described herein can contribute to assessing asubject's risk of developing a disorder associated with cholesterol,such as a cardiovascular disorder.

It should be appreciated that in assays described herein a detectablesignal, such as fluorescence, can be measured according to any methodknown in the art. In some aspects, samples are processed in multi-wellplates, such as 96-well or 384-well plates and fluorescence is measuredusing a plate reader. Systems associated with the invention can beconfigured such that fluorescence is measured and then correlated withoutputs such as the binding constant of a lipophilic or amphiphilicmolecule acceptor for a lipophilic or amphiphilic molecule.

As used herein, “binding constant” or “association constant” refers to amathematical constant that describes the binding affinity between twomolecules at equilibrium. It should be appreciated that methodsdescribed herein can also be used to measure dissociation constants.

Any type of detectable label can be compatible with aspects of theinvention. As used herein, a detectable label refers to a moiety, thepresence of which can be ascertained directly or indirectly. Generally,detection of a label involves an emission of energy by the label. Thelabel can be detected directly by its ability to emit and/or absorbphotons or other atomic particles of a particular wavelength (e.g.,radioactivity, luminescence, optical or electron density, etc.). A labelcan be detected indirectly by its ability to bind, recruit and, in somecases, cleave another moiety which itself may emit or absorb light of aparticular wavelength (e.g., epitope tag such as the FLAG epitope,enzyme tag such as horseradish peroxidase, etc.). An example of indirectdetection is the use of a first enzyme label which cleaves a substrateinto visible products. The label may be of a chemical, peptide ornucleic acid molecule nature although it is not so limited. Otherdetectable labels include radioactive isotopes such as P32 or H3,luminescent markers such as fluorochromes, optical or electron densitymarkers, etc., or epitope tags such as the FLAG epitope or the HAepitope, biotin, avidin, and enzyme tags such as horseradish peroxidase,bb-galactosidase, etc. The label may be bound to a peptide during orfollowing its synthesis.

Non-limiting examples of the types of labels that can be compatible withaspects of the claimed invention include enzymes, radioisotopes,fluorescent compounds, colloidal metals, chemiluminescent compounds, andbioluminescent compounds. Those of ordinary skill in the art will knowof other suitable labels for the molecules described herein, or will beable to ascertain such, using routine experimentation. Furthermore, thecoupling or conjugation of these labels to the molecules of theinvention can be performed using standard techniques common to those ofordinary skill in the art.

Another labeling technique consists of coupling molecules describedherein to low molecular weight haptens. These haptens can then bespecifically altered by means of a second reaction. For example, haptenssuch as biotin, can be used, which can react with avidin, ordinitrophenol, pyridoxal, or fluorescein, which can react with specificanti-hapten antibodies. Non-limiting examples of haptens includedigoxigenin, Alexa Fluor 488, Biotin-X SE, Biotin-XX SE, Biotin-XX SSE,BODIPY FL-X SE, BODIPY FL STP ester, Cascade Blue acetyl azide, Dansyl-XSE, DSB-X biotin SE, Lucifer yellow iodoacetamide, 5(6)-TAMRA-X SE,Rhodamine Red-X SE, Texas Red-X SE. Examples of corresponding antibodiesfor hapten conjugation included nut are not limited to Anti-Alexa Fluor488 dye, Anti-digoxigenin, Anti-biotin, Anti-BODIPY FL dye, Anti-CascadeBlue dye, Anti-DNP, anti-DNP-KLH, anti-biotin, Anti-fluorescein/OregonGreen dye, Anti-lucifer yellow, Anti-fluorescein/Oregon Green dye andAnti-tetramethylrhodamine.

A further category of detectable labels includes diagnostic and imaginglabels (generally referred to as in vivo detectable labels) such as forexample magnetic resonance imaging (MRI): Gd(DOTA); for nuclearmedicine: 201Tl, gamma-emitting radionuclide 99mTc; forpositron-emission tomography (PET): positron-emitting isotopes,(18)F-fluorodeoxyglucose ((18)FDG), (18)F-fluoride, copper-64,gadodiamide, and radioisotopes of Pb(II) such as 203Pb; 111In.

The conjugations or modifications described herein employ routinechemistry, which chemistry does not form a part of the invention andwhich chemistry is well known to those skilled in the art of chemistry.The use of protecting groups and known linkers such as mono- andhetero-bifunctional linkers are well documented in the literature andwill not be repeated here. As used herein, “conjugated” means twoentities stably bound to one another by any physiochemical means. Insome aspects, it is important that the nature of the attachment is suchthat it does not impair substantially the effectiveness of eitherentity. Any covalent or non-covalent linkage known to those of ordinaryskill in the art can be employed for conjugation. In some embodiments,covalent linkage is preferred. Noncovalent conjugation includeshydrophobic interactions, ionic interactions, high affinity interactionssuch as biotin-avidin and biotin-streptavidin complexation and otheraffinity interactions. Such means and methods of attachment are wellknown to those of ordinary skill in the art.

A variety of methods may be used to detect the label, depending on thenature of the label and other assay components. For example, the labelmay be detected while bound to the solid substrate or subsequent toseparation from the solid substrate. Labels may be directly detectedthrough optical or electron density, radioactive emissions, nonradiativeenergy transfers, etc. or indirectly detected with antibody conjugates,streptavidin-biotin conjugates, etc. Methods for detecting the labelsare well known in the art.

The term “secondary label” as used herein refers to moieties such asbiotin and various protein antigens that require the presence of asecond intermediate for production of a detectable signal. For biotin,the secondary intermediate may include streptavidin-enzyme conjugates.For antigen labels, secondary intermediates may include antibody-enzymeconjugates. Some fluorescent groups act as secondary labels because theytransfer energy to another group in the process of nonradiativefluorescent resonance energy transfer (FRET), and the second groupproduces the detected signal.

The term “mass-tag” as used herein refers to any moiety that is capableof being uniquely detected by virtue of its mass using mass spectrometry(MS) detection techniques. Examples of mass-tags include electrophorerelease tags such asN-[3-[4′-[(p-MethoxytetrafluorobenzŷoxyjphenylJ-S-methylglyceronylJisonipecoticAcid, 4′-[2, 3,5,6-Tetrafluoro-4-(pentafluorophenoxyl)]methylacetophenone, and their derivatives. The synthesis and utility of thesemass-tags is described in U.S. Pat. Nos. 4,650,750, 4,709,016,5,360,8191, 5,516,931, 5,602,273, 5,604,104, 5,610,020, and 5,650,270.Other examples of mass-tags include, but are not limited to,nucleotides, dideoxynucleotides, oligonucleotides of varying length andbase composition, oligopeptides, oligosaccharides, and other syntheticpolymers of varying length and monomer composition. A large variety oforganic molecules, both neutral and charged (biomolecules or syntheticcompounds) of an appropriate mass range (100-2000 Daltons) may also beused as mass- tags.

Primary labels include but are not limited to radioisotopes (e.g.tritium, 32P, 33P, 35S, 14C, 123I, 124I, 125I, or 131I), mass-tagsincluding, but not limited to, stable isotopes (e.g., 13C, 2H, 17O, 18O,15N, 19F, and 127I), positron emitting isotopes (e.g., 11C, 18F, 13N,124I, and 15O), and fluorescent labels are signal generating reportergroups which can be detected without further modifications. Detectablemoities may be analyzed by methods including, but not limited tofluorescence, positron emission tomography, SPECT medical imaging,chemiluminescence, electron-spin resonance, ultraviolet/visibleabsorbance spectroscopy, mass spectrometry, nuclear magnetic resonance,magnetic resonance, flow cytometry, autoradiography, scintillationcounting, phosphoimaging, and electrochemical methods.

The term “chemiluminescent group,” as used herein, refers to a groupwhich emits light as a result of a chemical reaction without theaddition of heat. By way of example, luminol(5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants likehydrogen peroxide (H2O2) in the presence of a base and a metal catalystto produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule whichabsorbs light of visible wavelengths, UV wavelengths or IR wavelengths.

The term “dye,” as used herein, refers to a soluble, coloring substancewhich contains a chromophore.

The term “electron dense group,” as used herein, refers to a group whichscatters electrons when irradiated with an electron beam. Such groupsinclude, but are not limited to, ammonium molybdate, bismuth subnitrate,cadmium iodide, carbohydrazide, ferric chloride hexahydrate,hexamethylene tetramine, indium trichloride anhydrous, lanthanumnitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate,periodic acid, phosphomolybdic acid, phosphotungstic acid, potassiumferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate,silver proteinate (Ag Assay: 8.0-8.5%) “Strong”, silvertetraphenylporphin (S-TPPS), sodium chloroaurate, sodium tungstate,thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranylnitrate, and vanadyl sulfate.

The term “energy transfer agent,” as used herein, refers to a moleculewhich either donates or accepts energy from another molecule. By way ofexample only, fluorescence resonance energy transfer (FRET) is adipole-dipole coupling process by which the excited-state energy of afluorescence donor molecule is non-radiatively transferred to anunexcited acceptor molecule which then fluorescently emits the donatedenergy at a longer wavelength. The term “moiety incorporating a heavyatom,” as used herein, refers to a group which incorporates an ion ofatom which is usually heavier than carbon. In some embodiments, suchions or atoms include, but are not limited to, silicon, tungsten, gold,lead, and uranium.

The term “photoaffinity label,” as used herein, refers to a label with agroup, which, upon exposure to light, forms a linkage with a moleculefor which the label has an affinity.

The term “photocaged moiety,” as used herein, refers to a group which,upon illumination at certain wavelengths, covalently or non-covalentlybinds other ions or molecules.

The term “photoisomerizable moiety,” as used herein, refers to a groupwherein upon illumination with light changes from one isomeric form toanother.

The term “radioactive moiety,” as used herein, refers to a group whosenuclei spontaneously give off nuclear radiation, such as alpha, beta, orgamma particles; wherein, alpha particles are helium nuclei, betaparticles are electrons, and gamma particles are high energy photons.

The term “spin label,” as used herein, refers to molecules which containan atom or a group of atoms exhibiting an unpaired electron spin (i.e. astable paramagnetic group) that in some embodiments are detected byelectron spin resonance spectroscopy and in other embodiments areattached to another molecule. Such spin-label molecules include, but arenot limited to, nitryl radicals and nitroxides, and in some embodimentsare single spin-labels or double spin-labels.

The term “quantum dots,” as used herein, refers to colloidalsemiconductor nanocrystals that in some embodiments are detected in thenear-infrared and have extremely high quantum yields (i.e., very brightupon modest illumination).

One of ordinary skill in the art will recognize that a detectable moietymay be attached to a provided compound via a suitable substituent. Asused herein, the term “suitable substituent” refers to a moiety that iscapable of covalent attachment to a detectable moiety. Such moieties arewell known to one of ordinary skill in the art and include groupscontaining, e.g., a carboxylate moiety, an amino moiety, a thiol moiety,or a hydroxyl moiety, to name but a few. It will be appreciated thatsuch moieties may be directly attached to a provided compound or via atethering moiety, such as a bivalent saturated or unsaturatedhydrocarbon chain. In some embodiments, detectable moieties are attachedto a provided compound via click chemistry. In some embodiments, suchmoieties are attached via a 1,3-cycloaddition of an azide with analkyne, optionally in the presence of a copper catalyst. Methods ofusing click chemistry are known in the art and include those describedby Rostovtsev et al., Angew. Chem. Int. Ed. 2002, 41, 2596-99 and Sun etal., Bioconjugate Chem., 2006, JJ, 52-57. In some embodiments, a clickready inhibitor moiety is provided and reacted with a click ready -T-R1moiety. As used herein, “click ready” refers to a moiety containing anazide or alkyne for use in a click chemistry reaction. In someembodiments, the click ready inhibitor moiety comprises an azide. Incertain embodiments, the click ready -T-R1 moiety comprises a strainedcyclooctyne for use in a copper-free click chemistry reaction (forexample, using methods described in Baskin et al., Proc. Natl. Acad.Sci. USA 2007, 104, 16793-16797).

Further aspects of the invention relate to using assays described hereinin screening methods for therapeutics. For example, therapeutics orpotential therapeutics can be added to samples within the assaysdescribed herein and the effect of the therapeutic or potentialtherapeutic on lipophilic or amphiphilic molecules, such as cholesterol,binding, transport or efflux can be measured.

Nanoparticles

Articles, compositions, kits, and methods relating to nanostructures,including those that can sequester molecules such as cholesterol, areprovided. Certain embodiments described herein include structures havinga core-shell type arrangement; for instance, a nanoparticle core may besurrounded by a shell including a material, such as a lipid bilayer,that can interact with cholesterol and/or other lipids. In someembodiments, the structures, when introduced into a subject, cansequester cholesterol and/or other lipids and remove them fromcirculation. Accordingly, the structures described herein may be used todiagnose, prevent, treat or manage certain diseases or bodilyconditions, especially those associated with abnormal lipid levels.

Certain structures described herein can mimic circulating lipoproteinssuch as high density lipoprotein (HDL) and low density lipoprotein(LDL), commonly referred to as “good” and “bad” cholesterol,respectively. One function of lipoproteins is to transport cholesteroland other lipids in the body in the aqueous blood, since these moleculesdo not normally dissolve in the blood. Lipoproteins are also responsiblefor a number of important pathologic functions such as atherosclerosis.These lipoproteins, and other similar circulating particles (e.g.,intermediate density lipoproteins, very low density lipoproteins, etc.),include nanostructures typically between 5 and 1000 nm. Each lipoproteinis unique with regard to its surface chemistry, size and composition.However, they also have in common an outer layer of phospholipids, aninner core of hydrophobic moieties (e.g., cholesteryl esters andtriglycerides), and a surface protein that identifies individuallipoprotein species and dictates physiology.

In some embodiments described herein, a core (e.g., a gold nanoparticle)can be used as a scaffold to template and direct the synthesis ofstructures of well defined size, shape, and surface chemistry that areamenable to a wide variety of further surface chemistry andtailorability. For example, a bottom-up, size-specific, lipoproteinsynthesis may be carried out by using a nanostructure core to support ashell including a lipid bilayer and/or other suitable components.

Articles and methods described herein involve the use of nanostructurescaffolds for controllable synthesis of structures with a high degree ofreproducibility and with the potential for massive scale-up. Theresulting structures may be stable in a variety of solvents, may havehigh in vivo circulation times, and may be relatively inexpensive tofabricate. Additionally, as lipids can be easily modified withcommercially available linker chemistries, the structures describedherein are amenable to further functionalization with potentialpharmacological agents and/or targeting/recognition agents such asantibodies, small molecules and proteins. Further advantages aredescribed in more detail below.

Nanostructures compatible with aspects of the invention are furtherdescribed in, and incorporated by reference from PCT/US2009/002540, thecontent of which is incorporated by reference herein in its entirety.

In embodiments in which the core is a nanostructure, the core includes asurface to which one or more components can be optionally attached. Forinstance, in some cases, core is a nanostructure surrounded by shell,which includes an inner surface and an outer surface. The shell may beformed, at least in part, of one or more components, such as a pluralityof lipids, which may optionally associate with one another and/or withsurface of the core. For example, components may be associated with thecore by being covalently attached to the core, physisorbed, chemisorbed,or attached to the core through ionic interactions, hydrophobic and/orhydrophilic interactions, electrostatic interactions, van der Waalsinteractions, or combinations thereof. In one particular embodiment, thecore includes a gold nanostructure and the shell is attached to the corethrough a gold-thiol bond.

Optionally, components can be crosslinked to one another. Crosslinkingof components of a shell can, for example, allow the control oftransport of species into the shell, or between an area exterior to theshell and an area interior of the shell. For example, relatively highamounts of crosslinking may allow certain small, but not large,molecules to pass into or through the shell, whereas relatively low orno crosslinking can allow larger molecules to pass into or through theshell. Additionally, the components forming the shell may be in the formof a monolayer or a multilayer, which can also facilitate or impede thetransport or sequestering of molecules. In one exemplary embodiment,shell includes a lipid bilayer that is arranged to sequestercholesterol, as described in more detail below.

It should be understood that a shell which surrounds a core need notcompletely surround the core, although such embodiments may be possible.For example, the shell may surround at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or at least 99% of the surface area ofa core. In some cases, the shell substantially surrounds a core. Inother cases, the shell completely surrounds a core. The components ofthe shell may be distributed evenly across a surface of the core in somecases, and unevenly in other cases. For example, the shell may includeportions (e.g., holes) that do not include any material in some cases.If desired, the shell may be designed to allow penetration and/ortransport of certain molecules and components into or out of the shell,but may prevent penetration and/or transport of other molecules andcomponents into or out of the shell. The ability of certain molecules topenetrate and/or be transported into and/or across a shell may dependon, for example, the packing density of the components forming the shelland the chemical and physical properties of the components forming theshell. As described herein, the shell may include one layer of material,or multilayers of materials in some embodiments.

Structure may also include one or more components such as proteins,nucleic acids, and bioactive agents which may optionally impartspecificity to the structure. One or more components may be associatedwith the core, the shell, or both; e.g., they may be associated withsurface of the core, inner surface of the shell, outer surface of theshell, and/or embedded in the shell. For example, one or more componentsmay be associated with the core, the shell, or both through covalentbonds, physisorption, chemisorption, or attached through ionicinteractions, hydrophobic and/or hydrophilic interactions, electrostaticinteractions, van der Waals interactions, or combinations thereof. Inone particular embodiment, shell is in the form of a lipoproteinassembly or structure which includes both proteins and lipids that arecovalently or non-covalently bound to one another. For example, theshell may be in the form of an apolipoprotein assembly that serves as anenzyme co-factor, receptor ligand, and/or lipid transfer carrier thatregulates the uptake of lipids. As described herein, the components ofstructure may be chosen such that the surface of the structure mimicsthe general surface composition of HDL, LDL, or other structures.

It should be understood that components and configurations other thanthose described herein may be suitable for certain structures andcompositions, and that not all of the components described arenecessarily present in some embodiments.

In some cases, core is hollow and therefore does not include ananostructure core. Thus, in some such and other embodiments, structureincludes a shell that can optionally allow components (e.g., bioactiveagents, cholesterol) to pass to and from core and an environment outsideof the shell. In contrast to certain existing hollow structures (e.g.,liposomes) which typically have a largest cross-sectional dimension ofgreater than about 100 nm due to the steric hindrance of the componentsforming the shell, structures having a hollow core (e.g., a partially orwholly hollow core) may be very small, e.g., having a largestcross-sectional dimension of less than about 100 nm, or even less thanabout 50 nm. For example, liposomes that include a lipid bilayercomprising phospholipids are difficult to fabricate having a size ofless than 100 nm since the phospholipids become limited sterically, thusmaking it difficult or impossible to form bilayered hollow structureswith small radii of curvature. Using the methods described herein,however, such and other structures having small radii of curvature canbe formed, as provided in more detail below.

In one set of embodiments, a structure, whether including ananostructure core or a hollow core, is constructed and arranged tosequester, transport, or exchange certain molecules to and/or from asubject or a biological sample. For instance, a structure, whenintroduced into a subject, may interact with one or more components inthe subject such as cells, tissues, organs, particles, fluids (e.g.,blood), and portions thereof. The interaction may take place, at leastin part, through the shell of a structure, and may involve, for example,the exchange of materials (e.g., proteins, peptides, polypeptides,nucleic acids, nutrients) from the one or more components of the subjectto a structure, and/or from a structure to the one or more components ofthe subject. In some such embodiments, the shell of a structure can bedesigned to include components with properties that allow favorableinteraction (e.g., binding, adsorption, transport) with the one or morematerials from the subject. For example, the shell may includecomponents having a certain hydrophobicity, hydrophilicity, surfacecharge, functional group, specificity for binding, and/or density tofacilitate particular interactions, as described in more detail below.In certain embodiments, one or more materials from a subject aresequestered by a structure, and a structure facilitates excretion,breakdown, and/or transport of the material. The excretion, breakdown,and/or transport of the material can lead to certain beneficial and/ortherapeutic effects. As such, the structures described herein can beused for the diagnosis, prevention, treatment or management of certaindiseases or bodily conditions.

In one particular set of embodiments, a structure, whether including ananostructure core or a hollow core, is constructed and arranged tosequester cholesterol (and/or other lipids). Without wishing to be boundby theory, it is hypothesized that a structure sequesters cholesterolthrough hydrophobic interactions with a hydrophobic layer (e.g., a lipidbilayer) of the structure. For example, in some cases, cholesterol canbind to a surface of the structure (e.g., to the outer surface of theshell) through hydrophobic interactions. In other cases, the cholesterolcan be transported from an outer surface of the shell to an innersurface of the shell and/or to the core of the structure. Thecholesterol can also be imbedded in the shell, e.g., between two layersof the shell. Optionally, a structure may include one or moreapolipoproteins (e.g., apoliprotein-A1), proteins, or peptides, whichcan facilitate the sequestering of cholesterol. A structure may alsosequester cholesterol by removing cholesterol and phospholipids from acell, or from other circulating lipoprotein species. cholesterolsequestered by a structure may be esterified enzymatically (e.g., bylecithin:acyl CoA transferase (LCAT)) to form a cholesteryl ester thatmay migrate towards the center of the structure. In the case of hollowcore embodiments, the cholesteryl ester may accumulate in the hollowcore.

Additionally, without wishing to be bound by theory, it is believed thatthe structures described herein can sequester cholesterol from highconcentrations of cholesterol (e.g., plaques) and transfer it to theliver directly or indirectly. For example, cholesterol may besequestered from areas of high concentrations of cholesterol (e.g.,plaques) by direct efflux of cholesterol from the plaque, or anycomponents of the plaque, into or onto the structures described herein.In some such embodiments, the cholesterol that is sequestered by thestructures is transported directly to the liver by the structures. Inother embodiments, other circulating lipoprotein species (e.g., LDL) mayparticipate in cholesterol exchange. For example, in some cases, freecholesterol or esterified cholesterol is transferred from otherlipoproteins to the structures described herein. In other cases, oncefree cholesterol or esterified cholesterol is sequestered by thestructures described herein, the cholesterol can be transferred from thestructures to the other lipoprotein species, which may ultimately end upin the liver. Thus, in such embodiments, the structures described hereincan augment reverse cholesterol transport indirectly. Furthermore, inthe case where free cholesterol or esterified cholesterol is sequesteredfrom the structures described herein to other lipoprotein species, thestructures may further sequester cholesterol from, for example, areas ofhigh cholesterol content, plaques, circulating lipoproteins, or otherphysiologic sites of high cholesterol concentration. It should beunderstood, however, that the structures described herein may removecholesterol and/or other molecules by other routes, such as throughurine, and the invention is not limited in this respect.

Accordingly, a structures may be used in the field of cardiovasculardisease for studying atherosclerosis and cholesterol transport, and,generally, to diagnose, prevent, treat or manage diseases or bodilyconditions associated with abnormal lipid levels, as described in moredetail below.

The amount of a molecule (e.g., cholesterol or other lipids) sequesteredby a structure and/or a composition described herein may depend on, forexample, the size of the structure, the biology and surface chemistry ofthe particle, as well as the method of administration. As such, a singlestructure described herein, which may be incorporated into apharmaceutical composition or other formulation, may be able tosequester any suitable number of a particular type of molecule (e.g.,lipids such as cholesterol; steroids such as estrogen, progesterone, andtestosterone; bile salts, etc.) during use, e.g., at least 2, at least5, at least 10, at least 20, at least 30, at least 50, at least 100, atleast 200, at least 500, at least 1,000, at least 2,000, at least 5,000,or at least 10,000 molecules, which may depend on the size (e.g.,surface area and/or volume) of the structure, the particularapplication, and the method of administration. In some cases, suchnumbers of molecules can be bound to the structure at one particularinstance.

In some cases, a single structure has a binding constant forcholesterol, K_(d), of, for example, less than or equal to about 100 μM,less than or equal to about 10 μM, less than or equal to about 1 μM,less than or equal to about 0.1 μM, less than or equal to about 10 nM,less than or equal to about 7 nM, less than or equal to about 5 nM, lessthan or equal to about 2 nM, less than or equal to about 1 nM, less thanor equal to about 0.1 nM, less than or equal to about 10 pM, less thanor equal to about 1 pM, less than or equal to about 0.1 pM, less than orequal to about 10 fM, or less than or equal to about 1 fM. Methods fordetermining the amount of cholesterol sequestered and binding constantsare provided in more detail below.

In certain embodiments, the molecules that are sequestered by thestructures described herein cause the structure to grow in size (e.g.,cross-sectional area, surface area and/or volume), e.g., depending onthe number of molecules sequestered. The molecules may associate with asurface of a structure, be imbedded in a shell of a structure, betransported to a core of the structure, or combinations thereof, asdescribed herein. As such, the size of a structure (e.g.,cross-sectional area, surface area and/or volume) can increase by atleast 5%, at least 10%, at least 20%, at least 30%, at least 50%, atleast 70%, or at least 100%, from a time prior to sequestration comparedto a time after/during sequestration in some embodiments.

It should be understood, however, that while many of the embodimentsherein are described in the context of sequestering cholesterol or otherlipids, the invention is not limited as such and the structures,compositions, kits, and methods described herein may be used tosequester other molecules and/or to prevent, treat, or manage otherdiseases or bodily conditions.

A core, whether being a nanostructure core or a hollow core, may haveany suitable shape and/or size. For instance, the core may besubstantially spherical, non-spherical, oval, rod-shaped, pyramidal,cube-like, disk-shaped, wire-like, or irregularly shaped. The core(e.g., a nanostructure core or a hollow core) may have a largestcross-sectional dimension (or, sometimes, a smallest cross-sectiondimension) of, for example, less than or equal to about 500 nm, lessthan or equal to about 250 nm, less than or equal to about 100 nm, lessthan or equal to about 75 nm, less than or equal to about 50 nm, lessthan or equal to about 40 nm, less than or equal to about 35 nm, lessthan or equal to about 30 nm, less than or equal to about 25 nm, lessthan or equal to about 20 nm, less than or equal to about 15 nm, or lessthan or equal to about 5 nm. In some cases, the core has an aspect ratioof greater than about 1:1, greater than 3:1, or greater than 5:1. Asused herein, “aspect ratio” refers to the ratio of a length to a width,where length and width measured perpendicular to one another, and thelength refers to the longest linearly measured dimension.

In embodiments in which a core includes a nanostructure core, thenanostructure core may be formed from any suitable material. Forinstance, in one embodiment, a nanostructure core comprises an inorganicmaterial. The inorganic material may include, for example, a metal(e.g., Ag, Au, Pt, Fe, Cr, Co, Ni, Cu, Zn, and other transition metals),a semiconductor (e.g., silicon, silicon compounds and alloys, cadmiumselenide, cadmium sulfide, indium arsenide, and indium phosphide), or aninsulator (e.g., ceramics such as silicon oxide). The inorganic materialmay be present in the core in any suitable amount, e.g., at least 1 wt%, 5 wt %, 10 wt %, 25 wt %, 50 wt %, 75 wt %, 90 wt %, or 99 wt %. Inone embodiment, the core is formed of 100 wt % inorganic material. Thenanostructure core may, in some cases, be in the form of a quantum dot,a carbon nanotube, a carbon nanowire, or a carbon nanorod. In somecases, the nanostructure core comprises, or is formed of, a materialthat is not of biological origin. In some embodiments, a nanostructureincludes one or more organic materials such as a synthetic polymerand/or a natural polymer. Examples of synthetic polymers includenon-degradable polymers such as polymethacrylate and degradable polymerssuch as polylactic acid, polyglycolic acid and copolymers thereof.Examples of natural polymers include hyaluronic acid, chitosan, andcollagen.

A structure, which may include a shell surrounding a core, may also haveany suitable shape and/or size. For instance, a structure may have ashape that is substantially spherical, oval, rod-shaped, pyramidal,cubed-like, disk-shaped, or irregularly shaped. The largestcross-sectional dimension (or, sometimes, a smallest cross-sectiondimension) of a structure may be, for example, less than or equal toabout 500 nm, less than or equal to about 250 nm, less than or equal toabout 100 nm, less than or equal to about 75 nm, less than or equal toabout 50 nm, less than or equal to about 40 nm, less than or equal toabout 35 nm, less than or equal to about 30 nm, less than or equal toabout 25 nm, less than or equal to about 20 nm, less than or equal toabout 15 nm, or less than or equal to about 5 nm. The structure may alsohave an aspect ratio substantially similar to the aspect ratio of thecore.

Furthermore, a shell of a structure can have any suitable thickness. Forexample, the thickness of a shell may be at least 10 Angstroms, at least0.1 nm, at least 1 nm, at least 2 nm, at least 5 nm, at least 7 nm, atleast 10 nm, at least 15 nm, at least 20 nm, at least 30 nm, at least 50nm, at least 100 nm, or at least 200 nm (e.g., from the inner surface tothe outer surface of the shell). In some cases, the thickness of a shellis less than 200 nm, less than 100 nm, less than 50 nm, less than 30 nm,less than 20 nm, less than 15 nm, less than 10 nm, less than 7 nm, lessthan 5 nm, less than 3 nm, less than 2 nm, or less than 1 nm (e.g., fromthe inner surface to the outer surface of the shell). Such thicknessesmay be determined prior to or after sequestration of molecules asdescribed herein.

Those of ordinary skill in the art are familiar with techniques todetermine sizes of structures and particles. Examples of suitabletechniques include dynamic light scattering (DLS) (e.g., using a MalvernZetasizer instrument), transmission electron microscopy, scanningelectron microscopy, electroresistance counting and laser diffraction.Other suitable techniques are known to those or ordinary skill in theart. Although many methods for determining sizes of nanostructures areknown, the sizes described herein (e.g., largest or smallestcross-sectional dimensions, thicknesses) refer to ones measured bydynamic light scattering.

The shell of a structure described herein may comprise any suitablematerial, such as a hydrophobic material, a hydrophilic material, and/oran amphiphilic material. Although the shell may include one or moreinorganic materials such as those listed above for the nanostructurecore, in many embodiments the shell includes an organic material such asa lipid or certain polymers. The components of the shell may be chosen,in some embodiments, to facilitate the sequestering of cholesterol orother molecules. For instance, cholesterol (or other sequesteredmolecules) may bind or otherwise associate with a surface of the shell,or the shell may include components that allow the cholesterol to beinternalized by the structure. Cholesterol (or other sequesteredmolecules) may also be embedded in a shell, within a layer or betweentwo layers forming the shell. The components of a shell may be charged,e.g., to impart a charge on the surface of the structure, or uncharged.

In one set of embodiments, a structure described herein or a portionthereof, such as a shell of a structure, includes one or more natural orsynthetic lipids or lipid analogs (i.e., lipophilic molecules). One ormore lipids and/or lipid analogues may form a single layer or amulti-layer (e.g., a bilayer) of a structure. In some instances wheremulti-layers are formed, the natural or synthetic lipids or lipidanalogs interdigitate (e.g., between different layers). Non-limitingexamples of natural or synthetic lipids or lipid analogs include fattyacyls, glycerolipids, glycerophospholipids, sphingolipids,saccharolipids and polyketides (derived from condensation of ketoacylsubunits), and sterol lipids and prenol lipids (derived fromcondensation of isoprene subunits).

In one particular set of embodiments, a structure described hereinincludes one or more phospholipids. The one or more phospholipids mayinclude, for example, phosphatidylcholine, phosphatidylglycerol,lecithin, β, γ-dipalmitoyl-α-lecithin, sphingomyelin,phosphatidylserine, phosphatidic acid,N-(2,3-di(9-(Z)-octadecenyloxy))-prop-1-yl-N,N,N-trimethylammoniumchloride, phosphatidylethanolamine, lysolecithin,lysophosphatidylethanolamine, phosphatidylinositol, cephalin,cardiolipin, cerebrosides, dicetylphosphate,dioleoylphosphatidylcholine, dipalmitoylphosphatidylcholine,dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol,palmitoyl-oleoyl-phosphatidylcholine, di-stearoyl-phosphatidylcholine,stearoyl-palmitoyl-phosphatidylcholine,di-palmitoyl-phosphatidylethanolamine,di-stearoyl-phosphatidylethanolamine, di-myrstoyl-phosphatidylserine,di-oleyl-phosphatidylcholine,1,2-dipalmitoyl-sn-glycero-3-phosphothioethanol, and combinationsthereof. In some cases, a shell (e.g., a bilayer) of a structureincludes 50-200 natural or synthetic lipids or lipid analogs (e.g.,phospholipids). For example, the shell may include less than about 500,less than about 400, less than about 300, less than about 200, or lessthan about 100 natural or synthetic lipids or lipid analogs (e.g.,phospholipids), e.g., depending on the size of the structure.

Non-phosphorus containing lipids may also be used such as stearylamine,docecylamine, acetyl palmitate, and fatty acid amides. In otherembodiments, other lipids such as fats, oils, waxes, cholesterol,sterols, fat-soluble vitamins (e.g., vitamins A, D, E and K), glycerides(e.g., monoglycerides, diglycerides, triglycerides) can be used to formportions of a structure described herein.

A portion of a structure described herein such as a shell or a surfaceof a nanostructure may optionally include one or more alkyl groups,e.g., an alkane-, alkene-, or alkyne-containing species, that optionallyimparts hydrophobicity to the structure. An “alkyl” group refers to asaturated aliphatic group, including a straight-chain alkyl group,branched-chain alkyl group, cycloalkyl (alicyclic) group, alkylsubstituted cycloalkyl group, and cycloalkyl substituted alkyl group.The alkyl group may have various carbon numbers, e.g., between C₂ andC₄₀, and in some embodiments may be greater than C₅, C₁₀, C₁₅, C₂₀, C₂₅,C₃₀, or C₃₅. In some embodiments, a straight chain or branched chainalkyl may have 30 or fewer carbon atoms in its backbone, and, in somecases, 20 or fewer. In some embodiments, a straight chain or branchedchain alkyl may have 12 or fewer carbon atoms in its backbone (e.g.,C₁-C₁₂ for straight chain, C₃-C₁₂ for branched chain), 6 or fewer, or 4or fewer. Likewise, cycloalkyls may have from 3-10 carbon atoms in theirring structure, or 5, 6 or 7 carbons in the ring structure. Examples ofalkyl groups include, but are not limited to, methyl, ethyl, propyl,isopropyl, cyclopropyl, butyl, isobutyl, tert-butyl, cyclobutyl, hexyl,cyclochexyl, and the like.

The alkyl group may include any suitable end group, e.g., a thiol group,an amino group (e.g., an unsubstituted or substituted amine), an amidegroup, an imine group, a carboxyl group, or a sulfate group, which may,for example, allow attachment of a ligand to a nanostructure coredirectly or via a linker. For example, where inert metals are used toform a nanostructure core, the alkyl species may include a thiol groupto form a metal-thiol bond. In some instances, the alkyl speciesincludes at least a second end group. For example, the species may bebound to a hydrophilic moiety such as polyethylene glycol. In otherembodiments, the second end group may be a reactive group that cancovalently attach to another functional group. In some instances, thesecond end group can participate in a ligand/receptor interaction (e.g.,biotin/streptavidin).

In some embodiments, the shell includes a polymer. For example, anamphiphilic polymer may be used. The polymer may be a diblock copolymer,a triblock copolymer, etc., e.g., where one block is a hydrophobicpolymer and another block is a hydrophilic polymer. For example, thepolymer may be a copolymer of an α-hydroxy acid (e.g., lactic acid) andpolyethylene glycol. In some cases, a shell includes a hydrophobicpolymer, such as polymers that may include certain acrylics, amides andimides, carbonates, dienes, esters, ethers, fluorocarbons, olefins,sytrenes, vinyl acetals, vinyl and vinylidene chlorides, vinyl esters,vinyl ethers and ketones, and vinylpyridine and vinylpyrrolidonespolymers. In other cases, a shell includes a hydrophilic polymer, suchas polymers including certain acrylics, amines, ethers, styrenes, vinylacids, and vinyl alcohols. The polymer may be charged or uncharged. Asnoted herein, the particular components of the shell can be chosen so asto impart certain functionality to the structures.

Where a shell includes an amphiphilic material, the material can bearranged in any suitable manner with respect to the nanostructure coreand/or with each other. For instance, the amphiphilic material mayinclude a hydrophilic group that points towards the core and ahydrophobic group that extends away from the core, or, the amphiphilicmaterial may include a hydrophobic group that points towards the coreand a hydrophilic group that extends away from the core. Bilayers ofeach configuration can also be formed.

The structures described herein may also include one or more proteins,polypeptides and/or peptides (e.g., synthetic peptides, amphiphilicpeptides). In one set of embodiments, the structures include proteins,polypeptides and/or peptides that can increase the rate of cholesteroltransfer or the cholesterol-carrying capacity of the structures. The oneor more proteins or peptides may be associated with the core (e.g., asurface of the core or embedded in the core), the shell (e.g., an innerand/or outer surface of the shell, and/or embedded in the shell), orboth. Associations may include covalent or non-covalent interactions(e.g., hydrophobic and/or hydrophilic interactions, electrostaticinteractions, van der Waals interactions).

An example of a suitable protein that may associate with a structuredescribed herein is an apolipoprotein, such as apolipoprotein A (e.g.,apo A-I, apo A-II, apo A-IV, and apo A-V), apolipoprotein B (e.g., apoB48 and apo B100), apolipoprotein C (e.g., apo C-I, apo C-II, apo C-III,and apo C-IV), and apolipoproteins D, E, and H. Specifically, apo A₁,apo A₂, and apo E promote transfer of cholesterol and cholesteryl estersto the liver for metabolism and may be useful to include in structuresdescribed herein. Additionally or alternatively, a structure describedherein may include one or more peptide analogues of an apolipoprotein,such as one described above. A structure may include any suitable numberof, e.g., at least 1, 2, 3, 4, 5, 6, or 10, apolipoproteins or analoguesthereof. In certain embodiments, a structure includes 1-6apolipoproteins, similar to a naturally occurring HDL particle. Ofcourse, other proteins (e.g., non-apolipoproteins) can also be includedin structures described herein.

Optionally, one or more enzymes may also be associated with a structuredescribed herein. For example, lecithin-cholesterol acyltransferase isan enzyme which converts free cholesterol into cholesteryl ester (a morehydrophobic form of cholesterol). In naturally-occurring lipoproteins(e.g., HDL and LDL), cholesteryl ester is sequestered into the core ofthe lipoprotein, and causes the lipoprotein to change from a disk shapeto a spherical shape. Thus, structures described herein may includelecithin-cholesterol acyltransferase to mimic HDL and LDL structures.Other enzymes such as cholesteryl ester transfer protein (CETP) whichtransfers esterified cholesterol from HDL to LDL species may also beincluded.

In some cases, one or more bioactive agents are associated with astructure or a composition described herein. The one or more bioactiveagents may optionally be released from the structure or composition(e.g., long-term or short-term release). Bioactive agents includemolecules that affect a biological system and include, for exampleproteins, nucleic acids, therapeutic agents, vitamins and theirderivatives, viral fractions, lipopolysaccharides, bacterial fractionsand hormones. Other agents of interest may include chemotherapeuticagents.

In some embodiments, one or more nucleic acids is associated with astructure described herein. A nucleic acids includes any double strandor single strand deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)of variable length. Nucleic acids include sense and anti-sense strands.Nucleic acid analogs such as phosphorothioates, phosphoramidates,phosphonates analogs are also considered nucleic acids and may be used.Nucleic acids also include chromosomes and chromosomal fragments.

One or more sugar residues can optionally be associated with structuresdescribed herein.

It should be understood that the components described herein, such asthe lipids, phospholipids, alkyl groups, polymers, proteins,polypeptides, peptides, enzymes, bioactive agents, nucleic acids, andspecies for targeting described above, may be associated with astructure in any suitable manner and with any suitable portion of thestructure, e.g., the core, the shell, or both. For example, one or moresuch components may be associated with a surface of a core, an interiorof a core, an inner surface of a shell, an outer surface of a shell,and/or embedded in a shell. Furthermore, such components can be used, insome embodiments, to facilitate the sequestration, exchange and/ortransport of materials (e.g., proteins, peptides, polypeptides, nucleicacids, nutrients) from one or more components of a subject (e.g., cells,tissues, organs, particles, fluids (e.g., blood), and portions thereof)to a structure described herein, and/or from the structure to the one ormore components of the subject. In some cases, the components havechemical and/or physical properties that allow favorable interaction(e.g., binding, adsorption, transport) with the one or more materialsfrom the subject.

Additionally, the components described herein, such as the lipids,phospholipids, alkyl groups, polymers, proteins, polypeptides, peptides,enzymes, bioactive agents, and nucleic acids, may be associated with astructure described herein prior to administration to a biologicalsample and/or after administration to a biological sample. For example,in some cases a structure described herein includes a core and a shellwhich is administered, and the structure has a greater therapeuticeffect after sequestering one or more components (e.g., anapolipoprotein) from a biological sample. That is, the structure may usenatural components from the biological sample to increase efficacy ofthe structure after it has been administered.

In one aspect, methods of making structures described herein areprovided. In some embodiments, methods include providing a fluidcomprising a plurality of nanostructures (e.g., nanostructure cores) anda first solvent, as well as a fluid comprising a plurality of componentsand a second solvent. First solvent may be chosen such that itstabilizes nanostructures, preventing the nanostructures fromprecipitating out of solution. Second solvent may be chosen so as tosolubilize components. The first and second solvents may be miscible insome embodiments, and immiscible in other embodiments. In certainembodiments in which solvents and are miscible with one another, thesolvents may also be miscible with water. Such and other solvents may beuseful in a single-phase synthesis. Solvents that are miscible orslightly miscible with water are known to those or ordinary skill in theart and include, for example, alcohols (e.g., ethanol, propanol), THF,DMF and DMSO. Organic solvents that are immiscible with water can alsobe used (e.g., in two-phase synthesis).

When components are combined with nanostructures, a shell comprisingcomponents is formed on the surface. The shell can include a monolayerof components, however, in other embodiments, multi-layers can be formed(e.g., at least two or at least three layers). If additional componentsare desired, the components can be combined and the components mayassociate with at least a portion of shell. For example, a secondcomponent present in a third solvent may be combined with nanostructureto form a structure including a shell in the form of a bilayer. Thebilayer may form due to favorable interaction between components, whichmay be the same or different. In certain embodiments, componentsinterdigitate.

Optionally, all or a portion of nanostructure may be removed from anassembled structure to form a partially or wholly hollow core.Nanostructure can be removed from the structure by a variety of methods,which may depend on the particular material used to form nanostructure.For instance, where nanostructure is a metal (e.g., gold) nanoparticle,solvents that are known to dissolve certain metals, such as iodine,potassium cyanide, and strong acids (e.g., nitric acid), can be used toremove the nanostructure core. Accordingly, in some cases where the coreis formed of a metal (e.g., Au(0)), removal of the metal may includeoxidizing the metal to form a metal salt, e.g., Au(0) to Au⁺ and/orAu³⁺. Electrochemical and redox methods can also be used to remove allor portions of a core. In some cases, a portion, but not all of thenanostructure core is removed, e.g., such that the nanostructure core isnow more porous than before the removal step. In other cases, the coreis released from the shell without removing a portion of the core. Forexample, a shell that is bonded to a metal core via sulfur-metal bondscan be released from the core by using small molecules such asdithiothreitol (DTT), which can displace the bonds. A suitable solventor a chemical may be chosen such that it can remove at least portions ofa core material, and/or release the shell from the core, withoutnegatively affecting the shape and/or configuration of the shell, and/ordegrade (e.g., denature) the components of the shell.

In certain embodiments, components are cross linked with one anotherprior to removing all or a portion of the nanostructure core. Forexample, components may be thiolated ligands which cross link by formingdisulfide bonds. Any suitable method for cross linking can be used, suchas photo cross linking, chemical cross linking, and oxidation-reductionmethods, as known to those of ordinary skill in the art. The crosslinking step may help to stabilize shell in the same or a similarconfiguration as that achieved when associated with nanostructure. Incertain embodiments, cross linking of components is performed at thesame time as the removal of nanostructure to form a partially or whollyhollow structure.

A similar approach for removing all or a portion of nanostructure can beused to form structure, which includes a shell comprising a bilayer ofcomponents surrounding a hollow core.

In some cases, instead of forming multiple layers of components on ananostructure surface in separate steps, multi-layers can be formed in asingle step. For instance, components may be combined in a single phaseof a liquid, e.g., a liquid that solubilizes and/or stabilizes thecomponents and the nanostructures. Such a liquid may, in some cases,comprise water, or a solvent that is miscible with water. In some suchembodiments, at least a first layer including components and a secondlayer including components are formed by self-assembly. The first andsecond layers in such a process may, in some instances, be formedsubstantially simultaneously. Additional layers can also be formed bysuch a process. Each of the layers can include a single component, ormixtures of components. To facilitate formation of the layers, a portionof the liquid may be removed from the mixture, e.g., by applying heat toevaporate a solvent having a low boiling point.

The ratio of components and nanostructures can be tailored depending on,for example, the type of components and nanostructures, the solvent(s)used, and the method of fabrication of the structures. For instance, toobtain solubility in aqueous solution, a suitable ratio can be chosensuch that there is an ample amount of a component on the surface of thenanostructure so as to maintain water solubility. Thus, in certaininstances, if the concentration of a component is too low, thestructures will not be stable. Furthermore, if the ratio is too highwith certain components, certain undesirable structures may be formedinstead of stable monodisperse structures. Those of ordinary skill inthe art can determine suitable ratios by simple experimentation incombination with the description provided herein.

Furthermore, additional components such as proteins, nucleic acids,polymers, bioactive agents (e.g., cholesterol lowering agents) or otherstructures can be associated with the structures at any step. Forexample, in some embodiments additional structures are added at the sametime as addition of components, prior to the addition of components, orafter the addition of components.

Advantageously, using the methods described herein, liposome-likestructures having a hollow core (or at least a partially hollow core)can be formed in a size range that is unique to certain existingliposomes. For example, for many existing liposomes formed from aphospholipid bilayer and having a hollow core, the liposomes are largeenough (e.g., typically greater than about 100 nm in diameter) such thatthe phospholipid bilayer is capable of being formed. As one attempts tomake liposomes of smaller diameter, the packing of phospholipid moietiesbecomes limited sterically thus making it difficult or impossible toform bilayered liposomal structures with small radii of curvature (e.g.,smaller than about 100 nm in diameter). Methods described herein,however, can be used to form structures of smaller diameter (e.g.,structures having a largest cross-sectional dimension of less than about100 nm, or less than or equal to about 50 nm), since the use of ananostructure core as a template allows the arrangement of components ina shell that is dictated, at least in part, by the size and shape of thenanostructure core. Such methods can be used to make biologicallyrelevant structures having a surface chemistry that mimics certainmolecules such as HDL and LDL.

Additionally, because structures described herein can be formed by theuse of nanostructures that serve as a template, and because certainnanostructures can be provided (e.g., made or purchased) havingrelatively high uniformity in size, shape, and mass, the structuresdescribed herein may also have relatively high uniformity in size,shape, and mass. That is, a mixture of relatively uniform structures canbe formed, where the plurality of structures have a distribution ofcross-sectional dimensions such that no more than about 20%, 15%, 10%,or 5% of the structures have a cross-sectional dimension greater thanabout 20%, 15%, 10%, or 5% of the average cross-sectional dimension.Structures having relatively high uniformity are useful in certaincompositions and methods described herein.

Furthermore, the structures that are formed using methods describedherein may disperse in a liquid, instead of forming aggregates.Dispersions of structures described herein are useful in certaincompositions and methods described herein.

Those of ordinary skill in the art can choose appropriate components,such as those cited in, and incorporated by reference, fromPCT/US2009/002540, nanostructure cores, and solvents useful for theformation of structures described herein by, for example, knowing theparticular components and nanostructure cores that would lead tofavorable structures, the physical properties of the components,nanostructures and solvents, and/or by a simple screening test. Onesimple screening test may include adding components (and/ornanostructures) to a solvent and determining whether the component (ornanostructure) is soluble or stabilized in the solvent and/or reactswith or is negatively affected by the solvent. Other simple tests can beconducted by those of ordinary skill in the art.

In one set of embodiments, the structures, compositions and methodsdescribed herein are used to diagnose, prevent, treat or manage diseasesor bodily conditions associated with abnormal lipid levels. Forinstance, high density lipoprotein is a dynamic serum nanostructureprotective against the development of atherosclerosis and resultantillnesses such as heart disease and stroke. Furthermore, in certainembodiments, diagnosis, prevention, treatment or management of diseasesor bodily conditions associated with abnormal lipid levels may involveusing the structures, compositions and methods described herein.

Other diseases or bodily conditions associated with abnormal lipidlevels which could benefit from methods and/or compositions describedherein include, for example, atherosclerosis, phlebosclerosis or anyvenous condition in which deposits of plaques containing cholesterol orother material are formed within the intima or inner media of veins,acute coronary syndromes, angina including, stable angina, unstableangina, inflammation, sepsis, vascular inflammation, dermalinflammation, congestive heart failure, coronary heart disease (CHD),ventricular arrythmias, peripheral vascular disease, myocardialinfarction, onset of fatal myocardial infarction, non-fatal myocardialinfarction, ischemia, cardiovascular ischemia, transient ischemicattacks, ischemia unrelated to cardiovascular disease,ischemia-reperfusion injury, decreased need for revascularization,coagulation disorders, thrombocytopenia, deep vein thrombosis,pancreatitis, non-alcoholic steatohepatitis, diabetic neuropathy,retinopathy, painful diabetic neuropathy, claudication, psoriasis,critical limb ischemia, impotence, dyslipidemia, hyperlipidemia,hyperlipoproteinemia, hypoalphalipoproteinemia, hypertriglyceridemia,any stenotic condition leading to ischemic pathology, obesity, diabetesincluding both Type I and Type II, ichtyosis, stroke, vulnerableplaques, lower-limb ulceration, severe coronary ischemia, lymphomas,cataracts, endothelial dysfunction, xanthomas, end organ dysfunction,vascular disease, vascular disease that results from smoking anddiabetes, carotid and coronary artery disease, regress and shrinkestablished plaques, unstable plaques, vessel intima that is weak,unstable vessel intima, endothelial injury, endothelial damage as aresult of surgical procedures, morbidity associated with vasculardisease, ulcerations in the arterial lumen, restenosis as a result ofballoon angioplasty, protein storage diseases (e.g., Alzheimer'sdisease, prion disease), diseases of hemostasis (e.g., thrombosis,thrombophilia, disseminated intravascular coagulation, thrombocytopenia,heparin induced thrombocytopenia, thrombotic thrombocytopenic purpura,),rheumatic diseases (e.g., multiple sclerosis, systemic lupuserythematosis, sjogren's syndrome, polymyositis/dermatomyositis,scleroderma), neuroligical diseases (e.g., Parkinson's disease,Alzheimer's disease), and subindications thereof.

Further non-limiting examples of conditions associated with cholesterollevels include: Abetalipoproteinemia, Familial Dysbetalipoproteinemia,Familial Lecithin cholesterol Acyltransferase Deficiency, Familiallipoprotein Lipase Deficiency, Hyperlipoproteinemias, Hypolipidemia,Tangier Disease, Alzheimers, coronary sclerosis, high blood pressure,macular degeneration, mixed dyslipidemia, primary hypercholesterolemia

In some embodiments, the disease or bodily condition is a metabolicand/or degenerative disease. Several non-limiting examples include:Alzheimer's disease, amyotrophic lateral sclerosis, amyotrophic lateralsclerosis (ALS), angina pectoris, arginase deficiency, atherosclerosis,cachexia, cancer, carbamylphosphate synthase deficiency, carboxylasedefects, cataracts, chronic obstructive pulmonary disease, chronictraumatic encephalopathy, citrullinaemia, congenital heart disease,congenital lactic acidosis, congenital myopathy, coronary arterydisease, coronary heart disease, crginosuccinic aciduria, cystinosis,diabetes, diabetic nephropathy, diabetic neuropathy, diabeticretinopathy, dilated cardiomyopathy, erectile dysfunction, essentialtremor, facioscapulohumeral muscular dystrophy, familial cardiomyopathy,Friedreich's ataxia, galactosemia, Gierke's disease, glomerulosclerosis,glutaric acidemia type I & II, glutaric aciduria type I, glycogenosis ordextrinosis, GSD type II (acid maltase deficiency—Pompe's disease), GSDtype III (glycogen debrancher deficiency—Cori's disease or Forbe'sdisease), GSD type IV (glycogen branching enzyme deficiency—Andersendisease), GSD type V (muscle glycogen phosphorylase deficiency—McArdledisease), GSD type VI (liver phosphorylase deficiency—Hers's disease),GSD type VII (muscle phosphofructokinase deficiency—Tarui's disease),GSD type XI (glucose transporter deficiency—Fanconi-Bickel disease),heart disease, homocystinuria, Huntington's disease, hyperinsulinemia,hyperlipidemia, hypertrophic cardiomyopathy, impaired glucosemetabolism, inflammatory bowel disease (IBD), isovaleric acidemia,keratoconus, lactic acidosis (pyruvate dehydrogenase complex defects),Lewy body disease, macular degeneration, maple syrup urine disease,medium chain acyl CoA dehydrogenase (MCAD) deficiency, methylmalonicacidemia, mitochondrial myopathies, mitochondrial myopathy,mitochondrial respiratory chain defects, multiple system atrophy, musclewasting syndrome, myotonic muscular dystrophy, n-acetyl glutamatesynthetase deficiency, Niemann-Pick disease, non-ketotichyperglycinaemia, ornithine carbamyl transferase deficiency,osteoarthritis, osteoporosis, Parkinson's disease, phenylketonuria,premenstrual syndrome, progressive supranuclear palsy, propionicacidemia, prostatitis, restrictive cardiomyopathy, rheumatic heartdisease, rheumatoid arthritis, sarcopenia, spinal muscular atrophy,Tay-Sachs disease, type 2 diabetes, tyrosinaemia (Type I), ulcerativecolitis, vascular restenosis, coronary heart disease.

In one particular embodiment, structures, compositions and methodsdescribed herein are used for assessing a subject's risk foratherosclerosis. In some embodiments, assays described herein can alsobe used to screen for agents to treat atherosclerosis. Treatingatherosclerosis may include performing a therapeutic intervention thatresults in reducing the cholesterol content of at least oneatherosclerotic plaque, or prophylactically inhibiting or preventing theformation or expansion of an atherosclerotic plaque. Generally, thevolume of the atherosclerotic plaque, and hence the degree ofobstruction of the vascular lumen, will also be reduced. The presentstructures, compositions and methods are particularly useful fortreating atherosclerotic lesions associated with familialhyperlipidemias.

Treatment of atherosclerosis may reduce the cholesterol content ofatherosclerotic plaques and/or the volume of atherosclerotic plaques.The cholesterol content may be reduced by, for example, at least10%-30%, at least 30%-50%, and in some instances at least 50%-85% ormore. The volume of the atherosclerotic plaques may also be reduced. Thereduction in plaque volume may be, for example, at least 5%-30%, oftenas much as 50%, and in some instances 75% or more. Methods ofdetermining the reduction of cholesterol content of atheroscleroticplaques and/or the volume of atherosclerotic plaques are known to thoseof ordinary skill in the art, and include intravascular ultrasound andmagnetic resonance imaging.

In another embodiment, structures, compositions and methods describedherein are used for assessing a subject's risk of having a vascular or acardiovascular condition or assessing whether the subject is at risk ofdeveloping a cardiovascular condition. Vascular conditions areconditions that involve the blood vessels (arteries and veins).Cardiovascular conditions are conditions that involve the heart and theblood vessels associated with the heart. Examples of vascular conditionsinclude diabetic retinopathy, diabetic nephropathy, renal fibrosis,hypertension, atherosclerosis, arteriosclerosis, atherosclerotic plaque,atherosclerotic plaque rupture, cerebrovascular accident (stroke),transient ischemic attack (TIA), peripheral artery disease, arterialocclusive disease, vascular aneurysm, ischemia, ischemic ulcer, heartvalve stenosis, heart valve regurgitation and intermittent claudication.Examples of cardiovascular conditions include coronary artery disease,ischemic cardiomyopathy, myocardial ischemia, and ischemic orpost-myocardial ischemia revascularization.

Structures, compositions and methods described herein can also be usedfor assessing a subject's risk of developing a cardiovascular condition.The degree of risk of a cardiovascular condition depends on themultitude and the severity or the magnitude of the risk factors that thesubject has. Risk charts and prediction algorithms are available forassessing the risk of cardiovascular conditions in a human subject basedon the presence and severity of risk factors. One commonly usedalgorithm for assessing the risk of a cardiovascular condition in ahuman subject based on the presence and severity of risk factors is theFramingham Heart Study risk prediction score. A human subject is at anelevated risk of having a cardiovascular condition if the subject's10-year calculated Framingham Heart Study risk score is greater than10%. Another method for assessing the risk of a cardiovascular event ina human subject is a global risk score that incorporates a measurementof a level of a marker of systemic inflammation, such as CRP, into theFramingham Heart Study risk prediction score. Other methods of assessingthe risk of a cardiovascular event in a human subject include coronarycalcium scanning, cardiac magnetic resonance imaging, and/or magneticresonance angiography.

The structures, compositions and methods described herein may also beuseful for prophylactic treatments.

Hyperlipidemias may also be assessed by the compositions and methodsdescribed herein.

In certain embodiments, structures and compositions described herein areused in a method involving the determination of a disease or conditionof a subject or biological sample. For instance, a method may includeintroducing a composition comprising a plurality of structures describedherein to a biological sample (e.g., in vitro), and exposing theplurality of nanostructures and/or the subject or biological sample totesting conditions that can determine a disease or condition of thesubject or biological sample.

It should be understood that any suitable structures described hereincan be used in such methods, including, for example, structures having ananostructure core comprising an inorganic material and a shellsubstantially surrounding and attached to the nanostructure core. Insome cases, such structures are adapted to sequester cholesterol. Inother cases, the structures are a marker for a disease or bodilycondition.

In other embodiments, a composition is introduced to a subject or abiological sample, and the structures of the composition and/or thesubject or biological sample are exposed to assay conditions that candetermine a disease or condition of the subject or biological sample. Atleast a portion of the structures may be retrieved from the subject orbiological sample and an assay may be performed with the structuresretrieved. The structures may be assayed for the amount and/or type ofmolecules bound to or otherwise sequestered by the structures. Forexample, in one set of embodiments, a competition assay is performed,e.g., where labeled cholesterol is added and displacement of cholesterolis monitored. The more measured uptake of labeled cholesterol, the lessbound un-labeled free cholesterol is present. This can be done, forexample, after a composition comprising the structures described hereinare administered to a subject or a biological sample, and the structuresare subsequently retrieved from the subject or biological sample. Thismethod can be used, for example, where the structures are to be used asa diagnostic agent to see how much cholesterol (unlabeled) it hassequestered in a subject or biological sample.

Other methods can also be used to determine the amount of cholesterolsequestered by structures described herein. In some cases, labeledcholesterol (e.g., fluorescently-labeled cholesterol such asNBD-cholesterol, BODIPY-cholesterol or radioactive cholesterol) can beused. Labeled cholesterol can be added to the structures either in vivoor in vitro. By adding structures without labeled cholesterol andmeasuring the fluorescence increase upon binding, one can calculate thebinding constant of labeled cholesterol to the structure. In addition,to remove the cholesterol from the structure, one can dissolve theparticle (e.g., KCN) and then measure the resultant fluorescence insolution. Comparing to standard curve can allow determination of thenumber of cholesterol molecules per particle. Other methods such asorganic extraction and quantitative mass spectrometry can also be usedto calculate amount of cholesterol sequestered by one or more structuresdescribed herein.

Further aspects of the invention relate to kits for measuring bindingbetween a cholesterol acceptor and cholesterol. Kits can comprise one ormore types of fluorescently labeled cholesterol analogs and one or moretypes of lapidated gold nanoparticles. Kits can further compriseinstructions for use.

The kits described herein may also contain one or more containers, whichcan contain components such as the structures, signaling entities,and/or biomolecules as described. The kits also may contain instructionsfor mixing, diluting, and/or administrating the compounds. The kits alsocan include other containers with one or more solvents, surfactants,preservatives, and/or diluents (e.g., normal saline (0.9% NaCl), or 5%dextrose) as well as containers for mixing, diluting or administeringthe components to the sample or to the patient in need of suchtreatment.

The compositions of the kit may be provided as any suitable form, forexample, as liquid solutions or as dried powders. When the compositionprovided is a dry powder, the powder may be reconstituted by theaddition of a suitable solvent, which may also be provided. Inembodiments where liquid forms of the composition are used, the liquidform may be concentrated or ready to use. The solvent will depend on theparticular inventive structure and the mode of use or administration.Suitable solvents for compositions are well known and are available inthe literature.

The kit, in one set of embodiments, may comprise one or more containerssuch as vials, tubes, and the like, each of the containers comprisingone of the separate elements to be used in the method. For example, oneof the containers may comprise a positive control in the assay.Additionally, the kit may include containers for other components, forexample, buffers useful in the assay.

As used herein, a “subject” or a “patient” refers to any mammal (e.g., ahuman), for example, a mammal that may be susceptible to a disease orbodily condition such as a disease or bodily condition associated withabnormal lipid levels. Examples of subjects or patients include a human,a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, acat or a rodent such as a mouse, a rat, a hamster, or a guinea pig.Generally, the invention is directed toward use with humans. A subjectmay be a subject diagnosed with a certain disease or bodily condition orotherwise known to have a disease or bodily condition. In someembodiments, a subject may be diagnosed as, or known to be, at risk ofdeveloping a disease or bodily condition. In some embodiments, a subjectmay be diagnosed with, or otherwise known to have, a disease or bodilycondition associated with abnormal lipid levels, as described herein. Incertain embodiments, a subject may be selected for diagnosis and/ortreatment on the basis of a known disease or bodily condition in thesubject. In some embodiments, a subject may be selected for diagnosisand/or treatment on the basis of a suspected disease or bodily conditionin the subject. In some embodiments, the presence of an existing diseaseor bodily condition may be suspected, but not yet identified in asubject.

A “biological sample,” as used herein, is any cell, body tissue, or bodyfluid sample obtained from a subject. Non-limiting examples of bodyfluids include, for example, lymph, saliva, blood, urine, and the like.Samples of tissue and/or cells for use in the various methods describedherein can be obtained through standard methods including, but notlimited to, tissue biopsy, including punch biopsy and cell scraping,needle biopsy; or collection of blood or other bodily fluids byaspiration or other suitable methods.

The following examples are intended to illustrate certain embodiments ofthe present invention, but are not to be construed as limiting and donot exemplify the full scope of the invention.

EXAMPLES Example 1: Development of a Cell-Free Competition Assay toMeasure the Dissociation Constant of Cholesterol and Human Serum Using aBiomimetic Nanostructure Introduction

Reverse Cholesterol Transport (RCT) is a major function of high densitylipoproteins and may protect against atherosclerosis. Measurement ofcellular RCT improves assessment of cardiovascular risk, but currentmethods of measurement are limited in clinical applicability. Here, arapid, cell-free competition assay to measure the dissociation constantof human serum for cholesterol is described using a fluorescentlylabeled cholesterol analog and a biomimetic lipidated gold nanoparticle.Using this assay, the K_(D) of human serum as well as a high densitylipoprotein-enriched fraction of serum is reported. Because cholesterolbinding to serum cholesterol acceptors is a significant step in cellularRCT, this assay has widespread applications for assessing cardiovascularrisk.

An intriguing property of colloidal gold is that it has the ability toquench fluorescent signals when the fluorescent moeity is in closephysical proximity to the gold nanoparticle. Boron-dipyrromethene(BODIPY)-labelled cholesterol is a highly fluorescent molecule which weshow (vide infra) to be strongly quenched by AuNP-HDL in adose-dependent and titratable manner. Using assays described herein, itwas found that BODIPY-Cholesterol is reversibly bound to AuNP-HDL, andthat the addition of a cholesterol acceptor, (such as ApoB-depletedhuman serum, which contains HDL) leads to recovery of the fluorescentsignal in a dose-dependent manner. Furthermore, because of the stabilityof the fluorescent signal, using this method, the binding capacity andkinetics of cholesterol binding to both HDL-AuNP and the competitivenatural cholesterol acceptor can be ascertained. An effective K_(D) ofthe cholesterol acceptor added, such as human serum, can also becalculated. These parameters are fundamental biochemical parameters thatare highly related to the process of reverse cholesterol transport.Measuring these functional parameters in a clinical test is very usefulin determining a patient's functional capacity for reverse cholesteroltransport.

Materials and Methods Synthesis of Lipidated Nanoparticles

Citrate-stabilized colloidal gold nanoparticles at 5 nm diameter and 80nM concentration were purchased from Ted Pella, Redding, Calif.Phospholipids were purchased from Avanti Polar Lipids, Alabaster, Ala.,and 1 mM stock solutions of each were made. 100% Ethanol (Sigma Aldrich,St. Louis, Mo.) as well as lipid stocks were added to the goldnanoparticle such that the final concentration of ethanol was 20%, thecolloidal gold was 64 nM, and 250-fold excesses of each lipid werepresent. After a two hour room temperature incubation, the solutionswere purified by diafiltration with a KrosFlo Research II tangentialflow filtration system (Spectrum Laboratories, Rancho Dominguez,Calif.). The samples were first concentrated down to a minimum holdupvolume of approximately 5 ml, and were then exchanged with buffer(Ultrapure 18.2 MΩ water) continuously for at least 7 volumes (35 ml) toremove excess lipid. Concentration of the samples was then assessed byabsorbance spectroscopy, using the extinction coefficient of 5 nm goldcolloid of 9.696×10⁶. Prior to use in further experiments the lipidatednanoparticle stock was diluted with ultrapure water to a finalconcentration of 1.0 μM.

In some embodiments, HDL AuNP constructs were synthesized as follows.Briefly, an 80 nM solution of 5 nm colloidal gold was incubated with afive-fold molar excess of purified ApoA1 protein. Next 250-fold molarexcess of1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(3-(2-pyridyldithio)proprionate(PDP PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipidswere added and incubated with the ApoA1-Gold Nanoparticle for 2 hours in20% Ethanol. Finally, excess reagents and ethanol were purified from theparticles using a tangential flow filtration system, resulting in stableHDL AuNPs in water. The concentration of these particles was thendetermined using absorbance at 525 nM, the absorption maximum for 5 nmcolloidal gold.

Fluorescence Measurements

All fluorescence measurements were made using black 96-well plates(Costar, Tewksbury, Mass.) on a BioTek Synergy 2 plate readingfluorometer (Winooski, Vt.) using read settings as follows: Tungstenlamp; Excitation filter: 485/20 nm; Emission filter: 528/20 nm; Opticsposition: Top read 510 nm; Sensitivity: 100. All reactions were carriedout in a total volume of 200 μl of water at 37° C. BODIPY Cholesterolstock solution used in these experiments was 125 μM in 100% Ethanol.Stock solution was made stored at −20° C.; working stocks were madefresh by diluting a portion of the stock solution to 1.25 μM or 2.5 μMin water. Serum samples used in this experiment were collected incompliance with Institutional guidelines for human subjects research.

Cellular RCT Assay

A cellular RCT assay was performed by plating J774 mouse macrophages(ATCC, Bethesda, Md.) on day 1, labeling the cells with ³H-Cholesterolon day 2, upregulating membrane cholesterol transport proteins with acyclic AMP analog on day 3, and incubating for 4 hours the radiolabeledcells with a cholesterol acceptor solution, such as human serum, on day4.^(3,4) Percent efflux was calculated as specific radioactivity in theserum-containing media divided by total radioactivity in the cells andthe media.

Increased percent efflux to ApoB-depleted serum (vide infra), a serumfraction relatively enriched in HDL,⁴ associates with coronary diseasestatus even after adjusting for total amount of HDL.³ Thus, quantifyingthe function of HDL independent of HDL concentration leads to improveddiagnostics. The cellular RCT assay may be conceptualized as beingdependent upon both cellular interactions with lipoproteins, andlipoprotein interactions with cholesterol itself. Importantly, datademonstrate the cell-independent component contributes most to overallefflux.⁵ The method herein demonstrated, allows measurement ofcholesterol binding with serum cholesterol acceptors, independent of thecellular context, and provides a means to calculate K_(D) values.

In some embodiments, the RCT assay was performed as follows: A 250 uMstock solution of BODIPY-Cholesterol in 100% Ethanol was created. 5 ulof BODIPY stock solution was added to a well of a 96-well plate suitablefor fluorescence measurements. 15 ul of Ethanol and 80 ul of water wereadded. Baseline fluorescence was then recorded using a plate-readingfluorometer using the excitation and emission filters of 485/20 and528/20, respectively. 100 ul of HDL AuNP at a concentration of 500 nMwas then added and fluorescence was recorded at 2 minute intervals for30 minutes. An approximately 70% decline in fluorescence was observedowing to quenching of BODIPY-Cholesterol signal upon binding of themolecule to the HDL-AuNP construct. A volume of approximately 20 ul ofcholesterol acceptor, such as purified ApoA1, Human HDL, orApoB-depleted human serum, was added and fluorescence was recorded at 2minute intervals for 60 minutes. Depending on the amount and capacity ofthe cholesterol acceptor added, fluorescence recovery was observed asthe new (non-quenching) cholesterol acceptor competes forBODIPY-Cholesterol bound to and quenched by the HDL-AuNP construct. Theamount of fluorescence recovery for a given cholesterol acceptormonotonically increased as a function of the concentration of thatacceptor (FIG. 1).

Preparation of ApoB-depleted serum was completed as follows.Polyethylene glycol with an average molecular weight of 8000 (PEG 8000)was prepared as a 20% weight/volume solution in 200 mM glycine, pH 7.4.Serum and PEG solution were mixed in a 10:4 ratio and incubated at roomtemperature for 20 minutes, then centrifuged at 12,700×g for 30 minutesat 4 degrees Celsius to pellet the ApoB fraction. The supernatant wasessentially free of ApoB containing lipoproteins, including low densitylipoprotein (LDL), and was therefore relatively enriched in HDL, freeApoA1, and albumin, all of which are capable of binding cholesterol.

This assay technology may exquisitely calculate the ability of humanserum cholesterol acceptors (e.g. Apo A1, HDLs) to efflux cholesterol ina non-radioactive and non-cell based assay. Furthermore, the assay canbe automated, scaled, is inexpensive, rapid, and easy. There are nocomparable tests on the market.

Results and Discussion

Methods described herein are based on a fluorescence-based competitionassay that harnesses the quenching capacity of the gold core of abiomimetic lipid construct to measure the K_(D) of cholesterol fromserum and fractions thereof. Data presented herein demonstrate that thisassay is capable of rapidly and quantitatively measuring cholesterolbinding to biomimetic nanostructures. Using assays developed herein,interrogation of cholesterol binding to complex biological matrices(e.g., human serum and serum HDL fractions) is quantitative, rapid, andcan be automated for high-throughput screening. Finally, the assay canbe easily adapted for applications where cholesterol binding, transport,or metabolism (or other similar fluorescently-tagged analytes), is ofinterest.

In order to study cholesterol binding, lipidated nanoparticles (NPs)were synthesized using a 5 nm diameter gold nanoparticle template core(FIG. 4).⁷ Gold nanoparticles (Ted Pella, 80 nM) were incubated with a250-fold excess each of1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[3-(2-pyridyldithio)propionate](PDP PE) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in 20%ethanol (v/v). Incubation took place for two hours at room temperature.The NP conjugates were purified from unreacted lipids by usingtangential flow filtration (Spectrum Laboratories, Rancho Dominguez,Calif.). Conjugates were characterized as previously described.⁷

To measure cholesterol binding, NPs were titrated into a solution with250 nM of fluorescent cholesterol analog, 23-(dipyrrometheneborondifluoride)-24-norCholesterol (BODIPY-Cholesterol, Ex: 495 nm, Em: 507nm). Fluorescence quenching was measured as the BODIPY-Cholesterol boundto the surface of the NPs. Binding was modeled as a receptor (NP) withmultiple binding sites (n_(NP,chol)) with identical dissociationconstants (K_(D,NP)). Binding isotherms were fit to the quadratic formof the binding equilibrium¹¹ (Equation 1) using non-linear least-squaresregression to estimate the parameters K_(D,NP) and n_(NP,chol):

$\begin{matrix}{\lbrack{Chol}\rbrack_{f} = \frac{\begin{matrix}{{- \left( {n_{{NP},{Chol}} + K_{D,{NP}} - \lbrack{Chol}\rbrack_{T}} \right)} +} \\\sqrt{\left( {{n_{{NP},{Chol}}\lbrack{NP}\rbrack} + K_{D,{NP}} - \lbrack{Chol}\rbrack_{T}} \right) + {4K_{D}} - \lbrack{Chol}\rbrack_{T}}\end{matrix}}{2}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

Where [Chol]_(f) is the free concentration of BODIPY-Cholesterol and[Chol]_(T) is the total concentration of BODIPY-Cholesterol. The Klotzplot is shown in FIG. 2; regression analysis demonstrated the K_(D,NP)for NP-Cholesterol was 40±9 nM, while n_(NP,chol) was 16±1. These valuesfavorably correspond to those reported for similar NPs.⁷

To determine the effective K_(D) of serum (K_(D,Serum)) for cholesterol,competition assays between NP and serum fractions were performed. Twotypes of serum fraction were tested: (1) whole serum, and (2)ApoB-depleted serum. ApoB-depleted serum was prepared by incubation ofserum in a 10:4 ratio with a solution of 20% w/v polyethylene glycol ata MW of 8000 and 200 mM glycine, pH 7.4 for 20 minutes at roomtemperature, followed by centrifugation at 12,800×g for 30 minutes at 4°C. This leaves a serum supernatant depleted of LDL particles, andenriched in HDL particles, lipid poor ApoA1, and albumin, a relevantfraction for assessing reverse cholesterol transport.³

Equation 2 gives a standard form of the competition equilibrium relatingK_(D,Serum) to EC₅₀:

$\begin{matrix}{K_{D,{Serum}} = {\frac{K_{D,{NP}}}{n_{{NP},{Chol}}\lbrack{NP}\rbrack} \times {EC}_{50}}} & \left( {{Equation}\mspace{14mu} 2} \right)\end{matrix}$

EC₅₀ is the fraction of serum in the reaction needed to recoverhalf-maximal fluorescence. Serum or ApoB-depleted serum were titratedinto solutions containing 20 nM lipidated NP and 125 nMBODIPY-Cholesterol, and incubated at 37° C. for 4 hours, by which pointequilibrium was reached (FIG. 3). The EC₅₀ for whole serum was 1.1×10⁻³,and was 2.2×10⁻³ for ApoB-depleted serum, yielding an effective K_(D)for whole serum of 1.4×10⁻⁴ and an effective K_(D) for ApoB-depletedserum of 3.0×10⁻⁴ (dimensionless units).

Because Equation 2 relies on several simplifying assumptions regardingbinding site occupancy and free ligand concentration, further assessmentof the validity of K_(D,Serum) determinations was performed by solvingthe exact cubic form of the competition equilibrium algebraically, thenusing non-linear least-squares regression to determine K_(D) asdescribed herein. The effective K_(D) for whole serum by this method was1.4×10⁻⁴ and for ApoB-depleted serum was 3.1×10⁻⁴. Thus K_(D,Serum) asassessed by Equation 2 was in excellent agreement with determinations byfitting to the exact formula.

Using the method reported herein, it is possible to calculate the K_(D)of cholesterol on a molar basis to a given cholesterol acceptor such asfree ApoA1 or a pure fraction of HDL. Using a concentration of ˜34 μMHDL in serum,¹² the apparent K_(D) for cholesterol per mol HDL isapproximately 10 nM, which compares favorably to the K_(D) of NP forcholesterol.

Values for the K_(D) of cholesterol interactions with lipoproteins havenot previously been reported. However, the strength of the interactionreported here compares well to the strength of porphyrin interactionswith low density lipoprotein (K_(D)=20 nM), as measured by quenching ofporphyrin ring autofluoresence upon interaction with low densitylipoprotein.¹³ Thus this technique allows for calculation of previouslyunreported K_(D) values, and may be extended to further refineunderstanding of cholesterol binding equilibria with lipoproteinspecies. However for clinical applications, simple measurement of theK_(D) in rapidly attainable bulk matrices such as serum or ApoB-depletedserum may be sufficient or even desirable. Reporting K_(D) as anaffinity constant (K_(A)) yields, in this example, a K_(A) for wholeserum of 7100 and for ApoB-depleted serum of 3300, meaningful andreadily comprehensible integers with higher values meaning tighterbinding, features that make K_(A) particularly well-suited for directinterpretation and implementation in clinical practice. Finally, thismethod can be extended to ascertain binding properties of complexmatrices for other lipophilic fluorescently-labeled ligands such astestosterone or cortisol.

In summary, the synthesis of a lipid nanostructure has been reportedwhich allows for the measurement of K_(D) of cholesterol with serum andserum fractions. To our knowledge, K_(D) of serum for cholesterol hasnot been previously reported. Measurement of this parameter in variousfractions of serum will facilitate construction of more accurate modelsof cholesterol flux. Furthermore this method is rapid, straightforward,and automatable and therefore will find clinical utility in improvingprediction of patients at risk for pathologies dependent uponcholesterol overload, such as certain forms of heart disease.

Example 2: Calculations and Nonlinear Regression Analysis

$\begin{matrix}{\lbrack{Chol}\rbrack_{f} = \frac{\begin{matrix}{{- \left( {n_{{NP},{Chol}} + K_{D,{NP}} - \lbrack{Chol}\rbrack_{T}} \right)} +} \\\sqrt{\left( {{n_{{NP},{Chol}}\lbrack{NP}\rbrack} + K_{D,{NP}} - \lbrack{Chol}\rbrack_{T}} \right) + {4K_{D}} - \lbrack{Chol}\rbrack_{T}}\end{matrix}}{2}} & \left( {{Equation}\mspace{14mu} 1} \right)\end{matrix}$

Equation 1 was derived from algebraic manipulation of the followingfundamental equations:

$\begin{matrix}{\lbrack{Chol}\rbrack_{f} = {\lbrack{Chol}\rbrack_{T} + \lbrack{Chol}\rbrack_{{Bound},{NP}}}} & \left( {{Equation}\mspace{14mu} {S1}} \right) \\{K_{D,{NP}} = \frac{\lbrack{Chol}\rbrack_{f}\left( {{n_{{NP},{Chol}}\lbrack{NP}\rbrack} - \lbrack{Chol}\rbrack_{{Bound},{NP}}} \right)}{\lbrack{Chol}\rbrack_{{Bound},{NP}}}} & \left( {{Equation}\mspace{14mu} {S2}} \right)\end{matrix}$

Where [Chol]_(Bound,NP) is the concentration of cholesterol bound to thenanoparticle. Note that the term (n_(Np,Chol)[NP]−[Chol]_(Bound,NP))denotes the concentration of free cholesterol binding sites in thereaction.

Nonlinear regression analysis for Equation 1 was performed usingGraphPad Prism 6 (La Jolla, Calif.) for the parameters n_(NP,chol) andK_(D,NP) by inputting a custom equation in the non-linear regression fitpackage:

Y=((Ltotal−Kd−n*10̂X+(4*Ltotal*Kd+(−Ltotal+Kd+n*10̂X)̂2)̂0.5)/2)   (EquationS3)

Where Y is free cholesterol, X is log [NP], n is n_(NP,Chol), Ltotal is[Chol]_(T).

To test whether Equation 2 is valid under experimental conditions used,where binding site occupancy may be a concern, the exact cubic root ofthe following system of equations was found using Mathematica 9.0(Champaign, Ill.). These equations are given below in a format suitablefor direct programming into Mathematica.

kdnpeq:=kdnp==(cf)(nnp*npt−cbnp)/cbnp;

kdseq:=kds==(cf)(ns*st−cbs)/cbs;

cteq:=ct==cf+cbs+cbnp;

This system of equations defines the following terms:kdnp—Kd of the Nanoparticlecf—free cholesterol concentrationnnp—number of binding sites on the nanoparticlenpt—total amount of nanoparticle in the reactioncbnp—amount of cholesterol bound to the nanoparticle==>(nnp*npt−cbnp)thus equals free binding sites on the nanoparticle and accounts forbinding site depletion.

Similarly:

kds—Kd of Serumns—number of binding sites on serum. This is for completeness we model\this lumped sum parameter as 1 binding sitest—total amount of serumcbs—cholesterol bound to serum==>(ns*st−cbs) thus equals free bindingsites in serum and accounts for binding site depletion.

Finally:

ct=Total cholesterolcf=free cholesterolcbs=cholesterol bound to serumcbnp=cholesterol bound to nanoparticle.

The system has 3 equations, and thus two variables can be eliminated. cfand cbs were selected for elimination, as cbnp is the direct read ofquenching and the assay measurement. From prior assay determinations wekdnp and nnp are known. ct and npt can be controlled. st is manipulatedas the titrate. ns in this instance is set to 1. Thus, an equation canbe solved for which only needs kds to be fit as a parameter.

Eliminate[{kdnpeq, kdseq, cteq}, {cf, cbs}]Algebraically eliminates cf and cbs from the system of equations,yielding the cubic function:

ct (−cbnp kdnp+cbnp kds+kdnp nnp npt−2 kds nnp npt+(kds nnp̂2npt̂2)/cbnp)/cbnp)==−cbnp̂2 kdnp+cbnp kdnp̂2+cbnp̂2 kds−cbnp kdnp kds+cbnpkdnp nnp npt−2 cbnp kds nnp npt+kdnp kds nnp npt+kds nnp̂2 npt̂2−cbnp kdnpns st+kdnp nnp npt ns st

This is set to be an equation, cbnpeq, which is then solved for cbnp,which is essentially a direct readout of the assay.

Solve[cbnpeq, cbnp]Yields one real root:

{cbnp −> −((−ct kdnp − kdnp{circumflex over ( )}2 + ct kds + kdnp kds −kdnp nnp npt +  2 kds nnp npt + kdnp ns st)/( 3 (kdnp − kds))) −(2{circumflex over ( )}( 1/3) (−(−ct kdnp − kdnp{circumflex over ( )}2 +ct kds + kdnp kds − kdnp nnp npt + 2 kds nnp npt + kdnp nsst){circumflex over ( )}2 +  3 (kdnp − kds) (ct kdnp nnp npt − 2 ct kdsnnp npt − kdnp kds nnp npt − kds nnp{circumflex over ( )}2npt{circumflex over ( )}2 − kdnp nnp npt ns st)))/(3 (kdnp − kds) (2ct{circumflex over ( )}3 kdnp{circumflex over ( )}3 +  6 ct{circumflexover ( )}2 kdnp{circumflex over ( )}4 + 6 ct kdnp{circumflex over( )}5 + 2 kdnp{circumflex over ( )}6 − 6 ct{circumflex over ( )}3kdnp{circumflex over ( )}2 kds −  18 ct{circumflex over ( )}2kdnp{circumflex over ( )}3 kds − 18 ct kdnp{circumflex over ( )}4 kds −6 kdnp{circumflex over ( )}5 kds +  6 ct{circumflex over ( )}3 kdnpkds{circumflex over ( )}2 + 18 ct{circumflex over ( )}2 kdnp{circumflexover ( )}2 kds{circumflex over ( )}2 + 18 ct kdnp{circumflex over ( )}3kds{circumflex over ( )}2 +  6 kdnp{circumflex over ( )}4 kds{circumflexover ( )}2 − 2 ct{circumflex over ( )}3 kds{circumflex over ( )}3 − 6ct{circumflex over ( )}2 kdnp kds{circumflex over ( )}3 −  6 ctkdnp{circumflex over ( )}2 kds{circumflex over ( )}3 − 2 kdnp{circumflexover ( )}3 kds{circumflex over ( )}3 − 3 ct{circumflex over ( )}2kdnp{circumflex over ( )}3 nnp npt +  3 ct kdnp{circumflex over ( )}4nnp npt + 6 kdnp{circumflex over ( )}5 nnp npt + 12 ct{circumflex over( )}2 kdnp{circumflex over ( )}2 kds nnp npt −  3 ct kdnp{circumflexover ( )}3 kds nnp npt − 15 kdnp{circumflex over ( )}4 kds nnp npt −  15ct{circumflex over ( )}2 kdnp kds{circumflex over ( )}2 nnp npt − 3 ctkdnp{circumflex over ( )}2 kds{circumflex over ( )}2 nnp npt +  12kdnp{circumflex over ( )}3 kds{circumflex over ( )}2 nnp npt + 6ct{circumflex over ( )}2 kds{circumflex over ( )}3 nnp npt +  3 ct kdnpkds{circumflex over ( )}3 nnp npt − 3 kdnp{circumflex over ( )}2kds{circumflex over ( )}3 nnp npt −  3 ct kdnp{circumflex over ( )}3nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 6 kdnp{circumflexover ( )}4 nnp{circumflex over ( )}2 npt{circumflex over ( )}2 −  3 ctkdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}2 npt{circumflexover ( )}2 − 12 kdnp{circumflex over ( )}3 kds nnp{circumflex over ( )}2npt{circumflex over ( )}2 +  12 ct kdnp kds{circumflex over ( )}2nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 3 kdnp{circumflexover ( )}2 kds{circumflex over ( )}2 nnp{circumflex over ( )}2npt{circumflex over ( )}2 −  6 ct kds{circumflex over ( )}3nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 3 kdnpkds{circumflex over ( )}3 nnp{circumflex over ( )}2 npt{circumflex over( )}2 +  2 kdnp{circumflex over ( )}3 nnp{circumflex over ( )}3npt{circumflex over ( )}3 − 3 kdnp{circumflex over ( )}2 kdsnnp{circumflex over ( )}3 npt{circumflex over ( )}3 −  3 kdnpkds{circumflex over ( )}2 nnp{circumflex over ( )}3 npt{circumflex over( )}3 + 2 kds{circumflex over ( )}3 nnp{circumflex over ( )}3npt{circumflex over ( )}3 −  6 ct{circumflex over ( )}2 kdnp{circumflexover ( )}3 ns st − 12 ct kdnp{circumflex over ( )}4 ns st − 6kdnp{circumflex over ( )}5 ns st +  12 ct{circumflex over ( )}2kdnp{circumflex over ( )}2 kds ns st + 24 ct kdnp{circumflex over ( )}3kds ns st +  12 kdnp{circumflex over ( )}4 kds ns st − 6 ct{circumflexover ( )}2 kdnp kds{circumflex over ( )}2 ns st −  12 ct kdnp{circumflexover ( )}2 kds{circumflex over ( )}2 ns st − 6 kdnp{circumflex over( )}3 kds{circumflex over ( )}2 ns st +  6 ct kdnp{circumflex over ( )}3nnp npt ns st − 3 kdnp{circumflex over ( )}4 nnp npt ns st −  9 ctkdnp{circumflex over ( )}2 kds nnp npt ns st + 9 kdnp{circumflex over( )}3 kds nnp npt ns st +  3 ct kdnp kds{circumflex over ( )}2 nnp nptns st − 6 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2 nnp nptns st +  3 kdnp{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 ns st − 12 kdnp{circumflex over ( )}2 kdsnnp{circumflex over ( )}2 npt{circumflex over ( )}2 ns st +  3 kdnpkds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex over( )}2 ns st + 6 ct kdnp{circumflex over ( )}3 ns{circumflex over ( )}2st{circumflex over ( )}2 +  6 kdnp{circumflex over ( )}4 ns{circumflexover ( )}2 st{circumflex over ( )}2 − 6 ct kdnp{circumflex over ( )}2kds ns{circumflex over ( )}2 st{circumflex over ( )}2 −  6kdnp{circumflex over ( )}3 kds ns{circumflex over ( )}2 st{circumflexover ( )}2 − 3 kdnp{circumflex over ( )}3 nnp npt ns{circumflex over( )}2 st{circumflex over ( )}2 −  3 kdnp{circumflex over ( )}2 kds nnpnpt ns{circumflex over ( )}2 st{circumflex over ( )}2 − 2kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex over( )}3 +  □((2 ct{circumflex over ( )}3 kdnp{circumflex over ( )}3 + 6ct{circumflex over ( )}2 kdnp{circumflex over ( )}4 + 6 ctkdnp{circumflex over ( )}5 + 2 kdnp{circumflex over ( )}6 −  6ct{circumflex over ( )}3 kdnp{circumflex over ( )}2 kds − 18ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 kds − 18 ctkdnp{circumflex over ( )}4 kds −  6 kdnp{circumflex over ( )}5 kds + 6ct{circumflex over ( )}3 kdnp kds{circumflex over ( )}2 + 18ct{circumflex over ( )}2 kdnp{circumflex over ( )}2 kds{circumflex over( )}2 +  18 ct kdnp{circumflex over ( )}3 kds{circumflex over ( )}2 + 6kdnp{circumflex over ( )}4 kds{circumflex over ( )}2 − 2 ct{circumflexover ( )}3 kds{circumflex over ( )}3 −  6 ct{circumflex over ( )}2 kdnpkds{circumflex over ( )}3 − 6 ct kdnp{circumflex over ( )}2kds{circumflex over ( )}3 − 2 kdnp{circumflex over ( )}3 kds{circumflexover ( )}3 −  3 ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 nnpnpt + 3 ct kdnp{circumflex over ( )}4 nnp npt + 6 kdnp{circumflex over( )}5 nnp npt +  12 ct{circumflex over ( )}2 kdnp{circumflex over ( )}2kds nnp npt − 3 ct kdnp{circumflex over ( )}3 kds nnp npt −  15kdnp{circumflex over ( )}4 kds nnp npt − 15 ct{circumflex over ( )}2kdnp kds{circumflex over ( )}2 nnp npt −  3 ct kdnp{circumflex over( )}2 kds{circumflex over ( )}2 nnp npt + 12 kdnp{circumflex over ( )}3kds{circumflex over ( )}2 nnp npt +  6 ct{circumflex over ( )}2kds{circumflex over ( )}3 nnp npt + 3 ct kdnp kds{circumflex over ( )}3nnp npt −  3 kdnp{circumflex over ( )}2 kds{circumflex over ( )}3 nnpnpt − 3 ct kdnp{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 +  6 kdnp{circumflex over ( )}4 nnp{circumflexover ( )}2 npt{circumflex over ( )}2 − 3 ct kdnp{circumflex over ( )}2kds nnp{circumflex over ( )}2 npt{circumflex over ( )}2 −  12kdnp{circumflex over ( )}3 kds nnp{circumflex over ( )}2 npt{circumflexover ( )}2 + 12 ct kdnp kds{circumflex over ( )}2 nnp{circumflex over( )}2 npt{circumflex over ( )}2 +  3 kdnp{circumflex over ( )}2kds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex over( )}2 − 6 ct kds{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 +  3 kdnp kds{circumflex over ( )}3nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 2 kdnp{circumflexover ( )}3 nnp{circumflex over ( )}3 npt{circumflex over ( )}3 −  3kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}3 npt{circumflexover ( )}3 − 3 kdnp kds{circumflex over ( )}2 nnp{circumflex over ( )}3npt{circumflex over ( )}3 +  2 kds{circumflex over ( )}3 nnp{circumflexover ( )}3 npt{circumflex over ( )}3 − 6 ct{circumflex over ( )}2kdnp{circumflex over ( )}3 ns st − 12 ct kdnp{circumflex over ( )}4 nsst −  6 kdnp{circumflex over ( )}5 ns st + 12 ct{circumflex over ( )}2kdnp{circumflex over ( )}2 kds ns st +  24 ct kdnp{circumflex over ( )}3kds ns st + 12 kdnp{circumflex over ( )}4 kds ns st −  6 ct{circumflexover ( )}2 kdnp kds{circumflex over ( )}2 ns st − 12 ct kdnp{circumflexover ( )}2 kds{circumflex over ( )}2 ns st −  6 kdnp{circumflex over( )}3 kds{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3nnp npt ns st −  3 kdnp{circumflex over ( )}4 nnp npt ns st − 9 ctkdnp{circumflex over ( )}2 kds nnp npt ns st +  9 kdnp{circumflex over( )}3 kds nnp npt ns st + 3 ct kdnp kds{circumflex over ( )}2 nnp npt nsst −  6 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2 nnp npt nsst + 3 kdnp{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 ns st −  12 kdnp{circumflex over ( )}2 kdsnnp{circumflex over ( )}2 npt{circumflex over ( )}2 ns st +  3 kdnpkds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex over( )}2 ns st + 6 ct kdnp{circumflex over ( )}3 ns{circumflex over ( )}2st{circumflex over ( )}2 +  6 kdnp{circumflex over ( )}4 ns{circumflexover ( )}2 st{circumflex over ( )}2 − 6 ct kdnp{circumflex over ( )}2kds ns{circumflex over ( )}2 st{circumflex over ( )}2 −  6kdnp{circumflex over ( )}3 kds ns{circumflex over ( )}2 st{circumflexover ( )}2 − 3 kdnp{circumflex over ( )}3 nnp npt ns{circumflex over( )}2 st{circumflex over ( )}2 −  3 kdnp{circumflex over ( )}2 kds nnpnpt ns{circumflex over ( )}2 st{circumflex over ( )}2 − 2kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex over( )}3){circumflex over ( )}2 + 4 (−(−ct kdnp − kdnp{circumflex over( )}2 + ct kds + kdnp kds − kdnp nnp npt +  2 kds nnp npt + kdnp nsst){circumflex over ( )}2 +  3 (kdnp − kds) (ct kdnp nnp npt − 2 ct kdsnnp npt −  kdnp kds nnp npt − kds nnp{circumflex over ( )}2npt{circumflex over ( )}2 −  kdnp nnp npt ns st)){circumflex over( )}3)){circumflex over ( )}(1/3)) + 1/(3 2{circumflex over ( )}(  1/3)(kdnp − kds)) (2 ct{circumflex over ( )}3 kdnp{circumflex over ( )}3 + 6ct{circumflex over ( )}2 kdnp{circumflex over ( )}4 + 6 ctkdnp{circumflex over ( )}5 + 2 kdnp{circumflex over ( )}6 − 6ct{circumflex over ( )}3 kdnp{circumflex over ( )}2 kds − 18ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 kds − 18 ctkdnp{circumflex over ( )}4 kds − 6 kdnp{circumflex over ( )}5 kds + 6ct{circumflex over ( )}3 kdnp kds{circumflex over ( )}2 + 18ct{circumflex over ( )}2 kdnp{circumflex over ( )}2 kds{circumflex over( )}2 + 18 ct kdnp{circumflex over ( )}3 kds{circumflex over ( )}2 + 6kdnp{circumflex over ( )}4 kds{circumflex over ( )}2 − 2 ct{circumflexover ( )}3 kds{circumflex over ( )}3 − 6 ct{circumflex over ( )}2 kdnpkds{circumflex over ( )}3 − 6 ct kdnp{circumflex over ( )}2kds{circumflex over ( )}3 − 2 kdnp{circumflex over ( )}3 kds{circumflexover ( )}3 − 3 ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 nnpnpt + 3 ct kdnp{circumflex over ( )}4 nnp npt + 6 kdnp{circumflex over( )}5 nnp npt + 12 ct{circumflex over ( )}2 kdnp{circumflex over ( )}2kds nnp npt − 3 ct kdnp{circumflex over ( )}3 kds nnp npt − 15kdnp{circumflex over ( )}4 kds nnp npt − 15 ct{circumflex over ( )}2kdnp kds{circumflex over ( )}2 nnp npt − 3 ct kdnp{circumflex over ( )}2kds{circumflex over ( )}2 nnp npt + 12 kdnp{circumflex over ( )}3kds{circumflex over ( )}2 nnp npt + 6 ct{circumflex over ( )}2kds{circumflex over ( )}3 nnp npt + 3 ct kdnp kds{circumflex over ( )}3nnp npt − 3 kdnp{circumflex over ( )}2 kds{circumflex over ( )}3 nnp npt− 3 ct kdnp{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4 nnp{circumflexover ( )}2 npt{circumflex over ( )}2 − 3 ct kdnp{circumflex over ( )}2kds nnp{circumflex over ( )}2 npt{circumflex over ( )}2 − 12kdnp{circumflex over ( )}3 kds nnp{circumflex over ( )}2 npt{circumflexover ( )}2 + 12 ct kdnp kds{circumflex over ( )}2 nnp{circumflex over( )}2 npt{circumflex over ( )}2 + 3 kdnp{circumflex over ( )}2kds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex over( )}2 − 6 ct kds{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 + 3 kdnp kds{circumflex over ( )}3nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 2 kdnp{circumflexover ( )}3 nnp{circumflex over ( )}3 npt{circumflex over ( )}3 − 3kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}3 npt{circumflexover ( )}3 − 3 kdnp kds{circumflex over ( )}2 nnp{circumflex over ( )}3npt{circumflex over ( )}3 + 2 kds{circumflex over ( )}3 nnp{circumflexover ( )}3 npt{circumflex over ( )}3 − 6 ct{circumflex over ( )}2kdnp{circumflex over ( )}3 ns st − 12 ct kdnp{circumflex over ( )}4 nsst − 6 kdnp{circumflex over ( )}5 ns st + 12 ct{circumflex over ( )}2kdnp{circumflex over ( )}2 kds ns st + 24 ct kdnp{circumflex over ( )}3kds ns st + 12 kdnp{circumflex over ( )}4 kds ns st − 6 ct{circumflexover ( )}2 kdnp kds{circumflex over ( )}2 ns st − 12 ct kdnp{circumflexover ( )}2 kds{circumflex over ( )}2 ns st − 6 kdnp{circumflex over( )}3 kds{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3nnp npt ns st − 3 kdnp{circumflex over ( )}4 nnp npt ns st − 9 ctkdnp{circumflex over ( )}2 kds nnp npt ns st + 9 kdnp{circumflex over( )}3 kds nnp npt ns st + 3 ct kdnp kds{circumflex over ( )}2 nnp npt nsst − 6 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2 nnp npt nsst + 3 kdnp{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 ns st − 12 kdnp{circumflex over ( )}2 kdsnnp{circumflex over ( )}2 npt{circumflex over ( )}2 ns st + 3 kdnpkds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex over( )}2 ns st + 6 ct kdnp{circumflex over ( )}3 ns{circumflex over ( )}2st{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4 ns{circumflexover ( )}2 st{circumflex over ( )}2 − 6 ct kdnp{circumflex over ( )}2kds ns{circumflex over ( )}2 st{circumflex over ( )}2 − 6kdnp{circumflex over ( )}3 kds ns{circumflex over ( )}2 st{circumflexover ( )}2 − 3 kdnp{circumflex over ( )}3 nnp npt ns{circumflex over( )}2 st{circumflex over ( )}2 − 3 kdnp{circumflex over ( )}2 kds nnpnpt ns{circumflex over ( )}2 st{circumflex over ( )}2 − 2kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex over( )}3 + □((2 ct{circumflex over ( )}3 kdnp{circumflex over ( )}3 + 6ct{circumflex over ( )}2 kdnp{circumflex over ( )}4 + 6 ctkdnp{circumflex over ( )}5 + 2 kdnp{circumflex over ( )}6 − 6ct{circumflex over ( )}3 kdnp{circumflex over ( )}2 kds − 18ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 kds − 18 ctkdnp{circumflex over ( )}4 kds − 6 kdnp{circumflex over ( )}5 kds + 6ct{circumflex over ( )}3 kdnp kds{circumflex over ( )}2 + 18ct{circumflex over ( )}2 kdnp{circumflex over ( )}2 kds{circumflex over( )}2 + 18 ct kdnp{circumflex over ( )}3 kds{circumflex over ( )}2 + 6kdnp{circumflex over ( )}4 kds{circumflex over ( )}2 − 2 ct{circumflexover ( )}3 kds{circumflex over ( )}3 − 6 ct{circumflex over ( )}2 kdnpkds{circumflex over ( )}3 − 6 ct kdnp{circumflex over ( )}2kds{circumflex over ( )}3 − 2 kdnp{circumflex over ( )}3 kds{circumflexover ( )}3 − 3 ct{circumflex over ( )}2 kdnp{circumflex over ( )}3 nnpnpt + 3 ct kdnp{circumflex over ( )}4 nnp npt + 6 kdnp{circumflex over( )}5 nnp npt + 12 ct{circumflex over ( )}2 kdnp{circumflex over ( )}2kds nnp npt − 3 ct kdnp{circumflex over ( )}3 kds nnp npt − 15kdnp{circumflex over ( )}4 kds nnp npt − 15 ct{circumflex over ( )}2kdnp kds{circumflex over ( )}2 nnp npt − 3 ct kdnp{circumflex over ( )}2kds{circumflex over ( )}2 nnp npt + 12 kdnp{circumflex over ( )}3kds{circumflex over ( )}2 nnp npt + 6 ct{circumflex over ( )}2kds{circumflex over ( )}3 nnp npt + 3 ct kdnp kds{circumflex over ( )}3nnp npt − 3 kdnp{circumflex over ( )}2 kds{circumflex over ( )}3 nnp npt− 3 ct kdnp{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4 nnp{circumflexover ( )}2 npt{circumflex over ( )}2 − 3 ct kdnp{circumflex over ( )}2kds nnp{circumflex over ( )}2 npt{circumflex over ( )}2 − 12kdnp{circumflex over ( )}3 kds nnp{circumflex over ( )}2 npt{circumflexover ( )}2 + 12 ct kdnp kds{circumflex over ( )}2 nnp{circumflex over( )}2 npt{circumflex over ( )}2 + 3 kdnp{circumflex over ( )}2kds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex over( )}2 − 6 ct kds{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 + 3 kdnp kds{circumflex over ( )}3nnp{circumflex over ( )}2 npt{circumflex over ( )}2 + 2 kdnp{circumflexover ( )}3 nnp{circumflex over ( )}3 npt{circumflex over ( )}3 − 3kdnp{circumflex over ( )}2 kds nnp{circumflex over ( )}3 npt{circumflexover ( )}3 − 3 kdnp kds{circumflex over ( )}2 nnp{circumflex over ( )}3npt{circumflex over ( )}3 + 2 kds{circumflex over ( )}3 nnp{circumflexover ( )}3 npt{circumflex over ( )}3 − 6 ct{circumflex over ( )}2kdnp{circumflex over ( )}3 ns st − 12 ct kdnp{circumflex over ( )}4 nsst − 6 kdnp{circumflex over ( )}5 ns st + 12 ct{circumflex over ( )}2kdnp{circumflex over ( )}2 kds ns st + 24 ct kdnp{circumflex over ( )}3kds ns st + 12 kdnp{circumflex over ( )}4 kds ns st − 6 ct{circumflexover ( )}2 kdnp kds{circumflex over ( )}2 ns st − 12 ct kdnp{circumflexover ( )}2 kds{circumflex over ( )}2 ns st − 6 kdnp{circumflex over( )}3 kds{circumflex over ( )}2 ns st + 6 ct kdnp{circumflex over ( )}3nnp npt ns st − 3 kdnp{circumflex over ( )}4 nnp npt ns st − 9 ctkdnp{circumflex over ( )}2 kds nnp npt ns st + 9 kdnp{circumflex over( )}3 kds nnp npt ns st + 3 ct kdnp kds{circumflex over ( )}2 nnp npt nsst − 6 kdnp{circumflex over ( )}2 kds{circumflex over ( )}2 nnp npt nsst + 3 kdnp{circumflex over ( )}3 nnp{circumflex over ( )}2npt{circumflex over ( )}2 ns st − 12 kdnp{circumflex over ( )}2 kdsnnp{circumflex over ( )}2 npt{circumflex over ( )}2 ns st + 3 kdnpkds{circumflex over ( )}2 nnp{circumflex over ( )}2 npt{circumflex over( )}2 ns st + 6 ct kdnp{circumflex over ( )}3 ns{circumflex over ( )}2st{circumflex over ( )}2 + 6 kdnp{circumflex over ( )}4 ns{circumflexover ( )}2 st{circumflex over ( )}2 − 6 ct kdnp{circumflex over ( )}2kds ns{circumflex over ( )}2 st{circumflex over ( )}2 − 6kdnp{circumflex over ( )}3 kds ns{circumflex over ( )}2 st{circumflexover ( )}2 − 3 kdnp{circumflex over ( )}3 nnp npt ns{circumflex over( )}2 st{circumflex over ( )}2 − 3 kdnp{circumflex over ( )}2 kds nnpnpt ns{circumflex over ( )}2 st{circumflex over ( )}2 − 2kdnp{circumflex over ( )}3 ns{circumflex over ( )}3 st{circumflex over( )}3){circumflex over ( )}2 +  4 (−(−ct kdnp − kdnp{circumflex over( )}2 + ct kds + kdnp kds − kdnp nnp npt + 2 kds nnp npt + kdnp nsst){circumflex over ( )}2 +  3 (kdnp − kds) (ct kdnp nnp npt − 2 ct kdsnnp npt − kdnp kds nnp npt − kds nnp{circumflex over ( )}2npt{circumflex over ( )}2 − kdnp nnp npt ns st)){circumflex over( )}3)){circumflex over ( )}(  1/3)}

To assess the performance of the function and how it performs with realparameters, the manipulate function is used (FIG. 5). For example:

Manipulate[LogLinearPlot[ct−(Cubic Root Argument noted above), {st,1*̂−5, 1*̂0}, PlotRange->{0, 250*10̂−9}], {ct, 1*̂−9, 500*̂−9,Appearance->“Labeled” }, {nnp, 1, 100, Appearance->“Labeled” }, {kdnp,0.1*̂−9, 7*̂−8, Appearance->“Labeled”}, {npt, 1*̂−9, 100*̂−9,Appearance->“Labeled” }, {kds, 0.0000001, 0.001, Appearance->“Labeled”}, {ns, 1, 10, Appearance->“Labeled” }]

This allows assessment of function performance as well as allows manualestimation of initial starting parameters for K_(D).

Next non-linear regression is performed. Although the root of thefunction is real, it contains imaginary parts which makes the built-inNonlinearModelFit function return an error because rounding errors leadto the return of a complex number with an extremely small imaginarypart, which disallows correct execution of the function.

Therefore, optimization was done semi-manually as follows using thefollowing standard method for least-squares error functions:

$\begin{matrix}{{Error} = {\sum\limits_{i}^{n}\left( {{observed}_{i} - {predicted}_{i}} \right)^{2}}} & \left( {{Equation}\mspace{14mu} {S4}} \right)\end{matrix}$

To find the minimum value of this function, K_(D) was stepped over arange of values until the minimum was found to a precision of 4 digitsfor K_(D). Initial choice of the range of K_(D) to test is guided byK_(D) values generated by standard EC₅₀ analysis, for example asimplemented in GraphPad Prism.

An example of the stepping argument which generates a list of Errors forincrementally different values of K_(D):

Do[Print[j] Print[Sum[(data[[i, 2]] − f3[250.*{circumflex over ( )}−9,15., 40.*{circumflex over ( )}−9, 20.*{circumflex over ( )}−9, j, 1,data[[i, 1]]]){circumflex over ( )}2, {i,  1, 48}]],  {j, 0.0001,0.0005, 0.000005}]

Where data is an array containing the experimental data with serumconcentration in column 1 and free cholesterol readings in column 2. Theargument {i, 1, 48} steps the function f3 (which contains the bindingequation solution) through the 48 data points in this data set. Theargument οj, 0.0001, 0.0005, 0.0000051 steps the value of K_(D) from0.0001 to 0.0005 in 0.000005 increments. A list of errors is printed andthe minimum error is found. Using this exact method, K_(D) for serum andApoB-depleted serum were found as reported herein. The values found byEquation 2 compared very favorably to these.

Example 3: Assay Measuring Patient Serum Affinity for CholesterolBinding

An experiment was performed to compare the methods of the invention withcurrently used methods. The data suggest a correlation between the twoassays. The assay of the invention performed consistently and in asimilar manner to the currently accepted assay.

In this experiment, 14 healthy donors donated 10 ml serum for furtherstudy. This serum was collected in the conventional manner using needlesand a vacutainer tube. Using this serum, a conventional cholesterolefflux assay using J774 macrophages and radiolabled cholesterol(³H-labelled cholesterol) was performed. (A. V. Khera, M. Cuchel, M. dela Llera-Moya, A. Rodrigues, M. F. Burke, K. Jafri, B. C. French, J. A.Phillips, M. L. Mucksavage, R. L. Wilensky, E. R. Mohler, G. H. Rothblatand D. J. Rader, The New England journal of medicine, 2011, 364,127-135.)

The radiolabelled cholesterol assay was performed as follows:

Sample Preparation for Cholesterol Efflux Assay.

Frozen serum aliquots from participants were gently thawed by roomtemperature incubation. 60 μl of serum was pipetted into amicrocentrifuge tube. To precipitate ApoB-containing lipoproteins fromthe sample, 24 μl of PEG solution (20% w/v of Polyethylene Glycol 8000(Sigma-Aldrich, Saint Louis, Mo.), 200 mM glycine, pH 7.4) was added andmixed by gently flicking the tube. After a 20 minute room temperatureincubation, the mixture was centrifuged at 12,700×g for 30 minutes at 4°C. To create the final efflux media, 50.4 μl of the supernatant was thenadded to 1750 μl MEM media with 25 mM HEPES buffer, 30 μM of cAMP analog(8-(4-Cholorphenylthio)adenosine 3′,5′-cyclic monophosphate sodium salt,Sigma-Aldrich, Saint Louis, Mo.), and 2 μg/ml Acyl-CoA Cholesterol AcylTransferase (ACAT) inhibitor (Sandoz 58-035, Sigma-Aldrich, Saint Louis,Mo.). This results in an efflux medium with 2.0% final concentration ofApo-B depleted serum, in sufficient volume for technical triplicates at500 μl each.

Efflux Assay.

Cholesterol efflux from cells to patient serum was measured using theassay developed by Rothblat, et al. J774 mouse macrophages were platedon 24-well plates at 150,000 cells per well on day 1. On day 2, thecells were radiolabeled with ³H-Cholesterol at 2 μCi/ml by incubatingeach well in 500 ul of RPMI media with 5% FBS and ³H-Cholesterol(Perkin-Elmer, Waltham, Mass.). After overnight incubation, toupregulate cholesterol efflux, cells were treated with 2 ug/ml ACATinhibitor, 300 μM cAMP analog in RPMI medium with 5% bovine serumalbumin for 18 hours. On day 4, the efflux assay was performed. Effluxmedium prepared from participant serum was placed on cells in technicaltriplicate at a volume of 500 μl per well. After four hours, 300 ul ofthe medium was aspirated and filtered to remove cellular debris. 250 ulof the filtered medium was assayed for radioactivity by scintillationcounting. Total radioactivity was assessed by counting radioactivity incells after isopropanol extraction. Percent efflux for each sample wascalculated as follows: (radioactivity in the medium afterefflux—radioactivity in blank medium lacking serum)/(total radioactivityin labeled cells). The percent efflux for the technical triplicates wasaveraged together. To normalize these efflux results across sample runs,a serum sample pool drawn from healthy volunteers was run with eachsample. Normalized efflux was calculated as percent efflux for thesample divided by percent efflux for the sample pool. This normalizedefflux value for each participant was then used for further associationand correlation analysis.

In this example, the normalized efflux assay was plotted on the x-axis,and could be seen to vary in magnitude among participants fromapproximately 0.8 to approximately 1.4, with a value of 1.0 beingequivalent to the efflux value of the pooled sample.

Nanoparticle Assay:

In parallel experiments, the nanoparticle-based sensor assay discussedhere was performed. In this particular experiment, nanoparticles at aconcentration of 30 uM, BODIPY-cholesterol at a concentration of 250 nM,and Apo-B depleted serum (prepared as described above) at a finalconcentration of 2% were mixed together in a total volume of 200 ul andallowed to incubate overnight in a 96-well plate. Mixtures were preparedin duplicate for each of the 14 participants. Background control wellslacking any serum (to measure maximum quenched signal in the presence ofnanoparticle) were also prepared. After overnight incubation,fluorescence was read using a 96-well plate reading fluorimeter, at anexcitation wavelength of 485 nm and an emission wavelength of 520 nm.

For each sample, from these fluorescence values was subtracted thebackground control fluorescence value to arrive at signal for eachserum, with higher signal reflecting enhanced ability of theparticipant's serum to compete with the nanoparticle forBODIPY-cholesterol. This value was normalized to thebackground-subtracted fluorescence value of the plasma pool, and plottedon the y-axis. It can be seen that the normalized nanoparticle-basedsensor assay value ranges from approximately 1.0 to 2.0. It can furtherbe seen from the graph that there is a correlation between the twoassays, with an r value of 0.39, and a slope of 0.51, and a p=0.17,which is close to statistical significance.

The analysis method in this example is different than the one describedabove. It can be readily appreciated that serum with a greater abilityto compete with nanoparticle for binding cholesterol will at any givenserum concentration be equal to that of serum with a lesser ability tocompete with nanoparticle for binding cholesterol, lead to greaterdequenching of serum BODIPY-cholesterol binding, and lead to a higherfluorescence value after correcting for background.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

REFERENCES

-   (1) Lusis, A. J. Nature 2000, 407, 233.-   (2) Rosenson, R. S.; Brewer, H. B., Jr.; Davidson, W. S.; Fayad, Z.    A.; Fuster, V.; Goldstein, J.; Hellerstein, M.; Jiang, X. C.;    Phillips, M. C.; Rader, D. J.; Remaley, A. T.; Rothblat, G. H.;    Tall, A. R.; Yvan-Charvet, L. Circulation 2012, 125, 1905.-   (3) Khera, A. V.; Cuchel, M.; de la Llera-Moya, M.; Rodrigues, A.;    Burke, M. F.; Jafri, K.; French, B. C.; Phillips, J. A.;    Mucksavage, M. L.; Wilensky, R. L.; Mohler, E. R.; Rothblat, G. H.;    Rader, D. J. N Engl J Med 2011, 364, 127.-   (4) de la Llera-Moya, M.; Drazul-Schrader, D.; Asztalos, B. F.;    Cuchel, M.; Rader, D. J.; Rothblat, G. H. Arteriosclerosis,    thrombosis, and vascular biology 2010, 30, 796.-   (5) Karlin, J. B.; Johnson, W. J.; Benedict, C. R.; Chacko, G. K.;    Phillips, M. C.; Rothblat, G. H. J Biol Chem 1987, 262, 12557.-   (6) Thaxton, C. S.; Daniel, W. L.; Giljohann, D. A.; Thomas, A. D.;    Mirkin, C. A. J Am Chem Soc 2009, 131, 1384.-   (7) Luthi, A. J.; Zhang, H.; Kim, D.; Giljohann, D. A.; Mirkin, C.    A.; Thaxton, C. S. ACS Nano 2012, 6, 276.-   (8) Dubertret, B.; Calame, M.; Libchaber, A. J. Nat. Biotechnol.    2001, 19, 365.-   (9) Seferos, D. S.; Giljohann, D. A.; Hill, H. D.; Prigodich, A. E.;    Mirkin, C. A. Journal of the American Chemical Society 2007, 129,    15477.-   (10) Demers, L. M.; Mirkin, C. A.; Mucic, R. C.; Reynolds, R. A.;    Letsinger, R. L.; Elghanian, R.; Viswanadham, G. Anal. Chem. 2000,    72, 5535.-   (11) Pollard, T. D. Mol Biol Cell 2010, 21, 4061.-   (12) Mackey, R. H.; Greenland, P.; Goff, D. C., Jr.; Lloyd-Jones,    D.; Sibley, C. T.; Mora, S. Journal of the American College of    Cardiology 2012, 60, 508.-   (13) Bonneau, S.; Vever-Bizet, C.; Morliere, P.; Maziere, J. C.;    Brault, D. Biophys J 2002, 83, 3470.

What is claimed is: 1.-9. (canceled)
 10. A kit for measuring anequilibrium constant of an acceptor for a lipophilic or amphiphilicmolecule, comprising: (a) the lipophilic or amphiphilic molecule havinga detectable signal; and (b) a structure, the structure comprising ananostructure core and a lipid layer surrounding and attached to thenanostructure core, wherein the structure quenches the signal of thelipophilic or amphiphilic molecule when the structure and the lipophilicor amphiphilic molecule are proximate.
 11. The kit of claim 10, furthercomprising instructions for use of the kit for measuring the equilibriumconstant of the acceptor for the lipophilic or amphiphilic molecule. 12.A system for measuring an equilibrium constant of an acceptor for alipophilic or amphiphilic molecule, comprising: (a) a sample, the samplecomprising: (i) a lipophilic or amphiphilic molecule, wherein themolecule can provide a detectable signal; (ii) a structure, thestructure comprising a nanostructure core and a lipid layer surroundingand attached to the nanostructure core, wherein the structure quenchesthe signal of the lipophilic or amphiphilic molecule when the structureand the lipophilic or amphiphilic molecule are proximate; (iii) anacceptor; and (b) a detector for measuring the signal.
 13. The system ofclaim 12, further comprising a device configured to calculate theequilibrium constant of the acceptor for the lipophilic or amphiphilicmolecule based on the detected signal.
 14. The system of claim 12,further comprising a radiation source configured to induce the signal.15.-32. (canceled)
 33. The kit of claim 10, wherein the equilibriumconstant is measured in a cell-free assay.
 34. The kit of claim 10,wherein the lipophilic or amphiphilic molecule comprises a steroid, alipopolysaccharide, a cholestane, cholane, pregnane, androstane, estraneor a derivative or analog thereof.
 35. The kit of claim 10, wherein thelipophilic or amphiphilic molecule comprises or consists ofBODIPY-cholesterol.
 36. The kit of claim 10, wherein the signal isfluorescence.
 37. The kit of claim 10, wherein the nanostructure core isan inorganic material.
 38. The kit of claim 10, wherein thenanostructure core is gold.
 39. The kit of claim 10, wherein thestructure further comprises an apolipoprotein bound to at least theouter surface of the lipid layer.
 40. The kit of claim 10, wherein theacceptor is a high-density lipoprotein (HDL).
 41. The system of claim12, wherein the sample is a biological sample and wherein the biologicalsample is not a cell.
 42. The system of claim 12, wherein the lipophilicor amphiphilic molecule comprises a steroid, a lipopolysaccharide, acholestane, cholane, pregnane, androstane, estrange, orBODIPY-cholesterol.
 43. The system of claim 12, wherein the signal isdetected by positron emission tomography, SPECT medical imaging,chemiluminescence, electron-spin resonance, ultraviolet/visibleabsorbance spectroscopy, mass spectrometry, nuclear magnetic resonance,magnetic resonance, flow cytometry, autoradiography, scintillationcounting, phosphoimaging, or an electrochemical method.
 44. The systemof claim 12, wherein the signal is fluorescence.
 45. The system of claim12, wherein the nanostructure core is gold.
 46. The system of claim 12,wherein the structure further comprises apolipoprotein bound to at leastthe outer surface of the lipid layer.
 47. The system of claim 12,wherein the acceptor is a high-density lipoprotein (HDL).